Zanamivir phosphonate congeners with anti-influenza  activity and determining oseltamivir susceptibility of influenza viruses

ABSTRACT

Methods and compositions for detection of drug resistant pathogens and treatment against infections thereof are provided. Methods for detection of oseltamivir-resistant influenza viruses by competitive binding assays utilizing non-oseltamivir influenza virus neuraminidase inhibitors and oseltamivir carboxylate are provided. Influenza virus neuraminidase inhibitors coupled to sensors and useful for employment in the methods of the invention are disclosed. Novel phosphonate compounds active as neuraminidase inhibitors against wild-type and oseltamivir-resistant influenza strains of H1N1, H5N1 and H3N2 viruses are disclosed. An enantioselective synthetic route to preparation of these phosphonate compounds via sialic acid is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/697,794, filed May 6, 2013, which is a U.S. national stage filingunder section 371 of International Patent Application Serial No.PCT/US2011/035982, filed May 10, 2011, and which claims priority of U.S.Provisional Patent Application Ser. No. 61/333,137, filed May 10, 2010and titled “Compositions and Methods for Determining OseltamivirSusceptibility of Influenza Viruses,” the contents of which areincorporated herein in their entirety by reference.

SEQUENCE LISTING

This application includes a Sequence Listing. The entire contends of theASCII text file created on Jul. 31, 2015, named G2112_01501_SL.txt,which is 2 kilobytes in size, and originally filed in parent U.S.application Ser. No. 13/697,794, is incorporated by reference in itsentirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to novel compounds that are effective againstinfluenza virus. The invention relates to novel phosphonate compoundsthat inhibit influenza virus neuraminidase from wild-type andoseltamivir-resistant strains of H1N1, H5N1 and H3N2 influenza viruses.The invention relates to detection of drug resistant pathogens. Inparticular, the invention relates to the detection ofostelamivir-resistant influenza viruses. More particularly, theinvention relates to the detection of ostelamivir-resistant influenzaviruses using novel compounds disclosed in the present invention.

BACKGROUND OF THE INVENTION

Outbreaks of influenza A virus continue to cause widespread morbidityand mortality worldwide. In the United States alone, an estimated 5 to20% of the population is infected by influenza A virus annually, causingapproximately 200,000 hospitalizations and 36,000 deaths. Theestablishment of comprehensive vaccination policies has been aneffective measure to limit influenza morbidity. However, the frequentgenetic drifting of the virus requires yearly reformulation of thevaccine, potentially leading to a mismatch between the viral strainpresent in the vaccine and that circulating.

Influenza A virus consists of 9 structural proteins and codesadditionally for two nonstructural NS1 proteins with regulatoryfunctions. The segmented nature of the viral genome allows the mechanismof genetic reassortment (exchange of genome segments) to take placeduring mixed infection of a cell with different viral strains. Theinfluenza A virus is classified into various subtypes depending on thedifferent hemagglutinin (HA) and neuraminidase (NA) viral proteinsdisplayed on their surface. Influenza A virus subtypes are identified bytwo viral surface glycoproteins, hemagglutinin (HA or H) andneuraminidase (NA or N). Each influenza virus subtype is identified byits combination of H and N proteins. There are 16 known HA subtypes and9 known NA subtypes.

Influenza virus is a negative-sense segmented RNA virus that can infectmany animal species including human. The replication of influenza genomeby the viral coded RNA dependent RNA polymerase is an error proneprocess generating progenies with varied genetic sequences at all times.Viable viruses with genetic alterations are designated “antigenic drift”mutants. The segmented nature of the viral genome and the possibility toinfect different animal species could produce “antigenic shift” mutants(P. K. Cheng et al., Emerg. Infect. Dis. 15, 966 (2009)). Underdesirable conditions, dominant variants may become prominent pathogensfor human or animals. The multi-step selection processes leading tomutant evolution are not completely understood (L. Cohen-Daniel et al.,J. Clin. Virol. 44, 138 (2009); R. Wagner, M. Matrosovich, H. D. Klenk,Rev. Med. Virol. 12, 159 (2002)). Whereas vaccines are often used forthe prevention of influenza virus infections, the most useful therapiesfor the treatment of influenza infections involve administration ofTamiflu® (the phosphate salt of oseltamivir ethyl ester, RocheLaboratories, Inc.) and Relenza® (zanamivir, Glaxo Wellcome, Inc.). (N.J. Cox, J. M. Hughes, N. Engl. J. Med. 341, 1387 (1999)). Oseltamivirand zanamivir are viral sialidase (neuraminidase) inhibitors thatprevent the release and dispersal of progeny virions within the mucosalsecretions and thereby reduce viral infectivity. Neuraminidase (NA), aglycoprotein expressed on the influenza virus surface, is essential forvirus replication and infectivity by breaking the linkage between theprogeny virus from the surface sialo-receptor of host cells. Thus,inhibition of NA by the structure-based strategy has been applied indiscovery of anti-influenza drugs.

Zanamivir (Relenza™) (von Itzstein, M. et al. Nature 1993, 363, 418.Dunn, C. J.; Goa, K. L. Drugs 1999, 58, 761.) is a popular drug for thetreatment of influenza. Tamiflu is a prodrug that is readily hydrolyzedby hepatic esterases to give the corresponding oseltamivir carboxylicacid as the active inhibitor to interact with three arginine residues(Arg118, Arg292 and Arg371) in the active site of viral neuraminidase(NA). (von Itzstein, M. et al. Nature 1993, 363, 418. Lew, W. et al.Curr. Med. Chem. 2000, 7, 663. Russell, R. J. et al. Nature 2006, 443,45.) Both oseltamivir and zanamivir inhibit influenza virus NA that isessential for virus propagation by cleaving the linkage between theprogeny virus from the surface sialo-receptor of host cells. The NAinhibitors are designed to have (oxa)cyclohexene scaffolds to mimic theoxonium transition-state in the enzymatic cleavage of sialic acid(N-acetylneuraminic acid), the outmost saccharide on the cell surfaceglycoprotein for binding with the active site of viral NA. Toaccommodate the binding with oseltamivir carboxylic acid, an induced fitof the NA to create a large hydrophobic pocket is needed for the3-pentyl side chain. (Collins, P. J., et al. Nature 2008, 453, 1258.) Incomparison, zanamivir is less susceptible to the newly evolved resistantviruses than oseltamivir phosphate. In the absence of the need forgenerating the hydrophobic binding pocket, the inhibition potency ofzanamivir to the NA mutant (e.g. the clinically relevant H274Y mutant)is unchanged.

Influenza A (H1N1) viruses bear a oseltamivir resistance conferringamino acid change of histidine to tyrosine at position 274 (H274Y) ofthe neuraminidase (NA) protein. The 2008 surge of the oseltamivirresistant H274Y mutants in seasonal H1N1 (A. Moscona, N. Engl. J. Med.360, 953 (2009)) was puzzling because the increases of these mutationsare not correlated to oseltamivir usage in many of the H274Y prevalentareas (J. Mossong et al., Antiviral Res. 84, 91 (2009); M. Jonges etal., Antiviral Res. 83, 290 (2009)). In addition, the H274Y oseltamivirresistant pandemic H1N1 (A. Gulland, Br. Med. J. 339, b4975 (2009)) andthe H5N1 mutants (Q. M. Le et al., Nature 437, 1108 (2005)) are reportedin patients suggesting that these mutants could impact influenza therapyoptions (I. Stephenson et al., Clin. Infect. Dis. 48, 389 (2009)).

Many derivatives of zanamivir have been prepared by modification at theglyceryl moiety scaffolds to mimic the oxonium transition-state in theenzymatic cleavage of sialic acid. The phosphonate group is generallyused as a bioisostere of carboxylate in drug design. (White, C. L. etal. J. Mol. Biol. 1995, 245, 623. Schug, K. A.; Lindner, W. Chem. Rev.2005, 105, 67. Streicher, H.; Busseb, H. Bioorg. Med. Chem. 2006, 14,1047.) In comparison with the carboxylate-guanidinium ion-pair, aphosphonate ion will exhibit stronger electrostatic interactions withthe guanidinium ion. Thus, the zanamivir phosphonate congener isexpected to have more potent against the neuraminidases of H1N1 and H5N1viruses, even the H274Y mutant. The enhanced affinity may beattributable to the strong electrostatic interactions of the phosphonategroup with the three arginine residues (Arg118, Arg292 and Arg371) inphysiological conditions.

Solution-phase neuraminidase inhibition assays normally use thefluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-acetyl-neuraminicacid, which is cleaved by neuraminidase to yield a fluorescent productthat can be quantified using a fluorometer (Potier et al., Anal.Biochem. 94:287-296 (1979)), however, this assay method is not amenableto a high-throughput format. In addition, due to the fast emergence ofresistant viral strains (see McKimm-Breschkin, J. L. Antiviral Res.2000, 47, 1-17), there remains a need to find new influenzaneuraminidase inhibitors.

SUMMARY OF THE INVENTION

The worldwide surge of oseltamivir resistant H1N1 influenza viruses in2008 has prompted the survey for resistant mutants and the need fordevelopment of quick tests for oseltamivir susceptibility. In the midstof the potentially prevalent oseltamivir resistant H1N1 viruses, thereis a need for a “point-of-care” test for quick assessment of theoseltamivir susceptibility of a patient's specimens in a doctor's officethat will benefit decisions regarding treatment options.

Novel phosphonate compounds are described. The compounds have activityas neuraminidase inhibitors against wild-type and oseltamivir-resistantstrains of H1N1, H5N1 and H3N2 influenza viruses. In some embodiments,the oseltamivir-resistant influenza strain comprises a H274Y mutation inneuraminidase. The present disclosure also provides an enantioselectivesynthetic route to the novel phosphonate compounds via sialic acid.

According to a feature of the present disclosure, a compound accordingto formula (I) is disclosed

wherein A is PO(OR)(OR5), wherein R and R⁵ are independently selectedfrom H, C1-C10 alkyl, aryl, aralkyl and X, wherein X is a cationiccounterion selected from the group consisting of ammonium, methylammonium, dimethylammonium, trimethylammonium, tetramethylammonium,ethanol-ammonium, dicyclohexylammonium, guanidinium, ethylenediammoniumcation, lithium cation, sodium cation, potassium cation, cesium cation,beryllium cation, magnesium cation, calcium cation, and zinc cation,wherein B is NHR⁶, NH₃ ^(+Y−), R⁶N(C═NH)NH₂ or R⁶N(C═NH₂ ⁺)NH₂Y⁻,wherein R⁶ represents hydrogen, C1-C₁₀ alkyl, aryl, or aralkyl, whereinY− is a anionic counterion selected from the group consisting ofchloride, bromide, iodide, acetate, trifluoroacetate, phosphate,diphosphate, nitrate, sulfate, benzenesulfonate, benzoate, salicylate,hydroxynaphthoate, fumarate, maleate, lactate, malate, succinate,tartrate, citrate, glutamate, gluconate, and stearate, wherein R¹ is CH₃or CF₃; wherein R² is H, C1-C10 alkyl or O═C—NHR⁷, wherein R⁷ representsa linker annexed with functional moiety such as biotin, fluorophore andanti-inflammatory agents, and wherein R³ and R⁴ are independentlyhydrogen, C1-C10 alkyl or O═C—R⁸, where R⁸ represents C1-C10 alkyl,aryl, or aralkyl.

A composition is disclosed comprising a therapeutically effective amountof formula (I) and a pharmaceutically acceptable excipient, wherein thecomposition is designed to be administered to an organism to inhibit theactivity of influenza virus neuraminidase.

According to a feature of the invention, a composition is disclosedcomprising a therapeutically effective amount of at least one of:

or at least one of:

or at least one of:

or at least one of:

According to a feature of the present disclosure, a process is disclosedfor making a composition of formula I the process comprising the stepsof:

(a) acetylating a chiral precursor sialic acid (2) to prepare anintermediate compound (3).

(b) treating intermediate compound (3) with diethyl trimethylsilylphosphite to form intermediate compound (4):

(c) treating intermediate (4) with N-bromosuccinimide under lightirradiation to give a bromo-substituted compound, which formsintermediate (5) in pyridine:

(d) treating intermediate compound (5) with trimethylsilyltrifluorosulfonate to form intermediate compound (6):

and (e) treating intermediate compound (6) with trimethylsilyl azide toform intermediate compound (7):

In some embodiments, the process further comprises treating intermediatecompound (7) with bromotrimethylsilane, with sodium methoxide and thenhydrogenating in sequence, to form compound (1a):

In some embodiments, the process further comprises treating intermediatecompound (7) with sodium ethoxide and then hydrogenating to formcompound (1c):

In some embodiments, the process further comprises hydrogenatingintermediate compound (7), and then reacting with1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea and Et3N to formintermediate compound (8):

In some embodiments, the process further comprises treating intermediatecompound (8) with bromotrimethylsilane, and then with sodium methoxideto form compound (1b):

In some embodiments, the process further comprises the step of treatingintermediate compound (8) with sodium ethoxide and then trifluoroaceticacid to form compound (1d):

In one aspect the invention relates to any product according to formula(I) produced by a process disclosed herein.

According to a feature of the present disclosure, a method is disclosedfor treating influenza infection, comprising providing a therapeuticallyeffective amount of a composition according to formula (I) to a subjectin need thereof.

According to certain aspects of the invention, the compound according toformula (I) is at least one of:

Another implementation of the invention provides a method of inhibitingthe activity of a neuraminidase comprising contacting said neuraminidasewith of any one of compounds (I). In one aspect, the neuraminidase is aninfluenza neuraminidase and the activity is inhibited in vivo. Inanother aspect, the activity is inhibited in vitro.

The invention relates to determining the drug susceptibility of apathogen by measuring binding of the drug and competitive inhibitorsthereof.

The invention relates to a method for determining the presence ofoseltamivir-resistant influenza virus, the method comprising the stepsof: (a) providing a sample suspected of comprising anoseltamivir-resistant influenza-virus or virus particle; (b) contactingthe sample with a binding molecule comprising a influenza neuraminidaserecognition unit (R) in the presence and absence of ostelamivircarboxylate (OC); and (c) determining a difference in binding of thebinding molecule to the influenza virus, or viral particle, in thepresence and absence of oseltamivir, wherein a lack of reduction inbinding level by the recognition unit (R) in the presence of oseltamiviras compared to the reduction in the level of binding when contacted witha ostelamivir-sensitive influenza virus control, indicates the presenceof an ostelamivir-resistant influenza virus in the sample. The bindingof the recognition unit (R) to the influenza virus is competitivelyinhibited by the concurrent binding by oseltamivir carboxylate.

In some embodiments, the oseltamivir-resistant influenza comprises amutation at amino acid position 274 of the neuraminidase (NA) protein ofinfluenza. In some embodiments, the mutation is H274Y.

In some aspects, the influenza virus containing sample is selected fromthe group consisting of mucus, saliva, respiratory secretion, throatwash, nasal wash, spinal fluid, sputum, urine, semen, sweat, feces,plasma, blood, bronchioalveolar fluid, vaginal fluid, tear fluid andtissue biopsy. In some embodiments, the influenza virus is obtained froma cell culture. In some embodiments, the cell culture is a Vero cellculture. In some embodiments, the influenza virus containing sample is acell infected with influenza virus.

In some aspects, the influenza virus containing sample is immobilized ona solid substrate. The solid substrate may be selected from the groupconsisting of a microwell, microtiter plates, silicon chips, glassslides, beads, microparticles, films, chromatography paper, membranes,bottles, dishes, slides, blotting material, filters, fibers, wovenfibers, shaped polymers, particles, dip-sticks, test tubes, chips,microchips, Langmuir Blodgett film, glass, germanium, silicon,(poly)tetrafluoroethylene, polystyrene, gallium arsenide, galliumphosphide, silicon oxide, silicon nitride, and combinations thereof. Insome embodiments, the influenza virus or virus particle is immobilizedon the solid substrate via the binding of a carbohydrate receptor to thehemagglutinin component of influenza virus.

In some aspects, the immobilization of the influenza virus or virusparticle to the solid substrate does not alter the binding of thebinding molecule or oseltamivir carboxylate to neuraminidase componentof the influenza virus or particle thereof.

In some embodiments, the recognition unit (R) is selected fromzanamivir, or tamiphosphor guanidine monoester, or phosphazanamivir orits monoester, and salts, esters and derivatives thereof. In someembodiments, the recognition unit (R) is coupled to a sensing unit (S)that is detectable. In some embodiments, the recognition unit (R) iscoupled to the sensing unit via a linker (L), such that the bindingmolecule has the structure R-L-S. In some embodiments, the linker (L)reduces the effect of the binding of the recognition unit to theinfluenza virus to the detection of the sensing unit (S).

The linker (L) may be selected from an aliphatic chain, a triazole, awater-soluble linker, and an ethylene glycol linker.

In some aspects, the sensing unit is directly or indirectly detectable.In some embodiments, the sensing unit is coupled to the detectablemoiety by a streptavidin-biotin interaction.

In some aspects, the sensing unit is a detectable moiety selected from afluorescent label, a gold label, an enzyme label, a radioactive label, aquantum dot label, and a protein label. In some embodiments, thedetectable moiety is detected by a signal selected from a luminescent,colorimetric, fluorimetric, or radioactive signal.

In some embodiments, the fluorescent label is selected from the groupconsisting of fluorescein, BODIPY, Alexa Fluor, Cy3, Cy5, Oregon Green,tetramethylrhodamine, Rhodamine Red, Texas Red, pyridyloxazole,benzoxadiazole derivatives, NBD halides, iodoacetamides, SBD; LuciferYellow, iodoacetamide; stilbene, coumarin, naphthalene, aziridine,dapoxyl, pyrene, bimanes, xanthene, cyanine, pyrene, phthalocyanine,phycobiliprotein, squarene dye, energy transfer dye combinations, andderivatives thereof.

In some embodiments, a biotin sensing unit is detectable by fluorescencelabeled streptavidin. In some embodiments, a fluorescence sensing unitis selected from fluorescein isothiocyanate (FITC), Alexa dyes andquantum dots. In some embodiments, the biotin sensing unit is detectableby streptavidin conjugated enzymes such as alkalian phosphatase,beta-galactosidase, or horse radish peroxidase. In some embodiments, atamiphosphor guanidine monoester is an ammonium salt thereof. In someembodiments, a phosphazanamivir is an ammonium salt thereof.

The disclosure relates to a compound that binds to the neuraminidase ofan influenza virus or particle thereof, wherein the binding iscompetitively inhibited by oseltamivir carboxylate (OC), the compoundcomprising the formula R-L-S, wherein: R is selected from zanamivir, ortamiphosphor guanidine monoester, or phosphazanamivir or its monoester,and salts, esters and derivatives thereof; L is optional, and selectedfrom a triazole linker, an aliphatic linker, or an ethylene glycollinker; and S is selected from (a) a moiety directly detectable by amethod selected from fluorescence, colorimety, luminescence andradioactive detection, or (b) an indirectly detectable moiety selectedfrom biotin and streptavidin conjugated reporting systems.

The disclosure relates to diagnostic kits, comprising a packagingmaterial and a composition for detecting presence of anoseltamivir-resistant influenza virus in a sample, wherein saidcomposition comprises a compound according to the disclosure. In someembodiments, the kit further comprises a label or package insertcomprising indications or directions for use of the kit for detection ofoseltamivir-resistant influenza virus in a sample.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a scheme for the synthesis of zanamivir phosphonatecongener and derivatives.

FIGS. 2A-2D show molecular models of compounds 1a (2A), 1b (2B),zanamivir (2C) and oseltamivir acid (2D).

FIG. 3 shows a scheme for synthesis of zanamivir-biotin conjugate (ZB,9).

FIG. 4 shows a scheme for synthesis of zanamivir-fluorescene conjugates15 and 17.

FIG. 5 shows a scheme for synthesis of tamiphosphor-biotin conjugate 24

FIG. 6 shows a scheme for synthesis of dizanamivir-biotin conjugate 26

FIGS. 7A-7B show determination of oseltamivir carboxylate (OC)susceptibility using immobilized influenza samples. (7A) Structures ofcompounds used in this study. (7B) OC susceptible (S) or OC resistant(R) WSN virus samples were immobilized in anti-HA coated microplatewells at 10⁵, 10⁴, or 10³ PFU per well. The immobilized viral samples intriplicate wells were incubated with 30 nM ZB for total binding or with30 nM ZB plus competing OC at 150 nM to measure OC resistant ZB binding.The bound viruses were further incubated with streptavidin conjugatedalkaline phosphatase followed by the chemiluminescent substrate tomeasure the relative luminescence units (RLU) increases due to thecatalysis by the bound alkaline phosphatase. The values of OC resistantZB binding were calculated as the ratios of RLU measured in the presenceof OC competition and those measured in the absence of OC. Conditionsresulting in significant binding reductions by competing OC are labeledwith “**” for p<0.001. The dashed line marked the 3% residual ZB bindingthat could not be inhibited completely with OC when high titer (10⁵ PFUper well) 274H viral samples were employed.

FIGS. 8A-8D show determination of OC susceptibility of clinicalinfluenza isolates collected in Taiwan in the years 2005-2009. SeasonalH1N1 collected in Taiwan in years 2005-2007 (8A), 2008 (8B), and 2009(8C) were used for OC susceptibility study. OC resistant ZB binding weremeasured and calculated as described in FIG. 1B. Binding values below 1%were presented as 1%. The dashed lines marked the 5% binding values wereused to call the susceptibility status of the testing viruses. (8D) OCsusceptibility of seven 2009 pandemic H1N1 isolates was measured in anidentical fashion except triplicate measurements were made on total andOC-resistant bindings. In addition, both total and OC resistant bindingsare shown.

FIGS. 9A-9D show the prototype “point-of-care” assay for OCsusceptibility determination of influenza viral samples. (9A) A PVDFmembrane with immobilized anti-HA antibody slots was used to absorbinfluenza samples that were pre-incubated with either 30 nM ZB or 30 nMZB plus 150 nM OC. In this study, the wild type WSN (274H) or the OCresistant WSN (274Y) mutant viruses at 10⁴, 10⁵, 10⁶ PFU per slot wereused. After blotting and washing, the membrane was treated withstreptavidin conjugated alkaline phosphatase and stained with BCIP/NBTto visually determine the OC susceptibility of the virus samples. (9B)Ten 2008 Taiwan seasonal H1N1 isolates, described in FIG. 8B, werevalidated using the membrane assay. (9C) Similarly, four pandemic H1N1viral strains tested in FIG. 8D were evaluated using the prototypeassay. (9D) OC competition of ZB binding was determined in several otherA-type or B-type influenza viruses using a modified method involvingdirect immobilization of the virus samples to a PVDF membrane without anantibody and then processed in an identical fashion.

FIG. 10 shows ZB (zanamivir biotin) binding at the surface of 293T cellsexpressing neuraminidase cDNA of A/Hanoi/30408/2005 H5N1. The 293T cellswere transfected with NA expression vector pcDNA3.1-NA by Lipofectamine™2000 (Invitrogen, Carlsbad, USA). After 48 hours, the transfected cellswere stained with 100 nM Zanamivir-Biotin (ZB) and further incubatedwith DyLight 488-conjugated streptavidin Immunofluorescence image wascaptured using a Leica TCS-SP5 laser scanning confocal microscope.

FIGS. 11A-11F show ZB binding of mixed 293 cells with varied contents ofcells expressing OC resistant neuraminidase. Recombinant 293T cellsexpressing OC sensitive (274H) and OC resistant (274Y) cells were mixedto different ratios as indicated: (11A) 274H:274Y=100:0, (11B)274H:274Y=999:1, (11C) 274H:274Y=99:1, (11D) 274H:274Y=95:5, (11E)274H:274Y=90:10, (11F) 274H:274Y=0:100. The mixed cells were treatedwith 10 nM ZB at room temperature for 1 hr followed by furtherdecoration with APC-conjugated streptavidin and analyzed by FACSCanto(Becton Dickinson) and FCS Express 3.0 software. The percentage of ZBlabeled cells were 11A, 83.95%; 11B, 84.4%; 11C, 84.62%; 11D, 86.07%;11E, 83.58%; and 11F, 75.1%.

FIGS. 12A-12F show oseltamivir carboxylate resistant ZB binding on mixed293T cells with varied contents of cells expressing OC resistantneuraminidase. The cells as in FIG. 11 were treated with 10 nM ZB andexcess OC at 300 nM and processed similarly. The percentage of ZBlabeled cells were 12A, 1.00%; 12B, 2.64%; 12C, 2.78%; 12D, 7.10%; 12E,12.41%; and 12F, 68.29%.

FIGS. 13A-13B show OC competition of ZB binding to influenza virusinfected MDCK cells. (13A) MDCK cells were infected with OC susceptible274H or OC resistant 274Y viruses. At 20 hr post-infection, the 274Hvirus infected cells were incubated with ZB at 10 nM (open triangle) or50 nM (open circle) in the presence of varied concentrations ofcompeting OC. Similarly, the 274Y infected cells were also incubatedwith 10 nM ZB (closed triangle) or 50 nM ZB (closed circle) with variedOC contents. Cells with bound ZB were further incubated withstreptavidin conjugated alkaline phosphatase to determine the relativeZB binding at different competing OC concentrations. (13B) Image of MDCKcells infected with OC susceptible (274H) or OC resistant (274Y) WSNviruses and incubated either with 30 nM ZB or 30 nM ZB plus 150 nM OCfollowed by incubation with streptavidin conjugated PE. The fluorescenceimages were captured using a laser driven plate reader exited andemitted at 488 and 575 nm, respectively.

FIG. 14 shows RABC assay of immobilized WSN viruses for estimating theOC resistant viral contents by ZB binding with competing OC. Mixed WSNvirus samples with varying 274H and 274Y virus contents were immobilizedin triplicates each in anti-HA coated microwells at 10⁵ PFU (triangles),10⁴ PFU (squares), and 10³ PFU (circles) per well. They were added with30 nM ZB and 150 nM OC for one hr followed by coupling with streptavidinconjugated alkaline phosphatase to determine the estimated percent OCresistant values that are plotted against the contents of 274Y used forthe experiment.

DETAILED DESCRIPTION OF THE INVENTION

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

Oseltamivir is an oral prodrug that is converted by endogenous esterasesto oseltamivir carboxylate (OC). Zanamivir works by binding to theactive site of the neuraminidase protein, rendering the influenza virusunable to escape its host cell and infect others. (Cyranoski D(September 2005) Nat. Medicine 11 (9): 909). It is also an inhibitor ofinfluenza virus replication in vitro and in vivo. Bioavailability ofzanamivir is 2% and it is usually administered by inhalation.

Recent reports on the drug resistant avian flu infections and the sideeffects in children receiving Tamiflu® treatments suggest that newchemical identities for neuraminidase inhibitors (NAIs) are needed forthe battle against the threat of the pandemic flu. The NA inhibitors aredesigned to have (oxa)cyclohexene scaffolds to mimic the oxoniumtransition-state in the enzymatic cleavage of sialic acid, (Russell etal., Nature 2006, 443:45). On hydrolysis by hepatic esterases, theactive carboxylate, oseltamivir is exposed to interact with threearginine residues (Arg118, Arg292 and Arg371) in the active site of NA.(Id.).

Synthesis of Zanamivir Phosphonate Congeners with Anti-InfluenzaActivity

The present disclosure provides a novel synthetic route to novelphosphonate congers of zanamivir. D-Sialic acid was utilized as a chiralprecursor for the synthesis of novel active neuraminidase inhibitors.Novel phosphonate congers exhibit better anti-flu activities thanzanamivir and oseltamivir by inhibiting the neuraminidases of thewild-type and H274Y mutant of H1N1 and H5N1 viruses.

In one implementation, the invention provides a novel synthetic methodto enantioselective synthesis of various zanamivir phosphonate congenersand the derivatives with reasonably high yields. The synthetic route isshown in FIG. 1. Reagents and steps described in FIG. 1 are as follows:

Step 1. Sialic acid (2) was treated with acetic anhydride in pyridine toafford the peracetylation intermediate, which was heated at 100° C. toinduce decarboxylation, giving compound 3. (Horn, E. J., et alCarbohydr. Res. 2008, 343, 936).

Step 2. Compound 3 was treated with trimethylsilyl diethyl phosphate asthe appropriate nucleophile to afford the phosphonate compound 4 as amixture of a and 3 anomers (2:3), which could be separated bychromatography and characterized by NMR spectral analyses.

Step 3. Phosphonates 4 (as an anomeric mixture) was irradiated withN-bromosuccinimide (NBS) in CH₂Cl₂ solution to undergo a radical typebromination, which was treated in situ with pyridine to afford aβ-elimination product 5, a phosphonate derivative of Neu5Ac2en (DANA).This synthetic method for 5 was much more efficient than a lengthyprocedure reported previously for its analogue of dimethyl phosphonate(Vasella, A.; Wyler, R. Helv. Chim. Acta 1991, 74, 451).

Step 4. In a media of acetic anhydride, acetic acid and concentratedH₂SO₄, compound 5 was converted into the oxazoline 6.

Step 5. The regio- and stereoselective ring-opening reaction ofoxazoline 6 with azidotrimethylsilane was carried out to afford an azidocompound 7 as the pivotal intermediate leading to compounds 1a, 1b, 1cand 1d.

Step 6. Compound 7 was converted to phosphonic acid 1a by a sequence ofthree reactions: removal of both ethyl groups from phosphonate 7 withbromotrimethylsilane, deacetylation using sodium methoxide in methanol,and selective reduction of the azido group to amine by hydrogenation inthe presence of Lindlar catalyst.

Alternatively, only one ethyl group was removed from phosphonate diester7 on treatment with sodium ethoxide in ethanol, giving phosphonatediester 1c after the subsequent reduction of the azido group.

Step 7. To introduce a guanidino substituent, the azido group incompound 7 was first reduced to give amine, which reacted with1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea in the presence ofmercury chloride and triethylamine to afford guanidine compound 8.

Step 8. Both ethyl groups in phosphonate 8 were removed bybromotrimethylsilane, and the concurrent removal of tert-butoxycarbonyl(Boc) groups was realized by workup with methanol due to the in situgenerated HBr. The zanamivir phosphonate (1b) was obtained bydeacetylation.

Alternatively, phosphonate diester 7 was converted to a monoester withsodium ethoxide, followed by removal of Boc groups with trifluoroaceticacid, to give compound 1d.

The compositions of this invention optionally comprise salts of thecompounds herein, especially pharmaceutically acceptable non-toxic saltscontaining, for example, Na⁺, Li⁺, K⁺, Ca⁺⁺ and Mg⁺⁺. Such salts mayinclude those derived by combination of appropriate cations such asalkali and alkaline earth metal ions or ammonium and quaternary aminoions with an acid anion moiety.

Metal salts are prepared by reacting the metal hydroxide with a compoundof this invention. Examples of metal salts which are prepared in thisway are salts containing Na⁺, Li⁺, K⁺.

In addition, salts may be formed from acid addition of certain organicand inorganic acids, e.g., HCl, HBr, H₂SO₄, or organic sulfonic acids,to basic centers, typically amines Finally, it is to be understood thatthe compositions herein comprise compounds of the invention in theirun-ionized, as well as zwitterionic form, and combinations withstoichiometric amounts of water as in hydrates. Another aspect of theinvention relates to methods of inhibiting the activity of neuraminidasecomprising the step of treating a sample suspected of containingneuraminidase with a compound of the invention.

The compounds of this invention are formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotonic. All formulations will optionally contain excipientssuch as those set forth in the “Handbook of Pharmaceutical Excipients”(1986), which is expressly incorporated by reference in its entirety.Excipients include ascorbic acid and other antioxidants, chelatingagents such as EDTA, carbohydrates such as dextrin,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations ranges from about pH 3 to about pH 11,but is ordinarily about pH 7 to pH 10.

One or more compounds of the invention (herein referred to as the activeingredients) are administered by any route appropriate to the conditionto be treated. Suitable routes include oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural), and the like. It will be appreciated that the preferred routemay vary with for example the condition of the recipient.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the inventioncomprise at least one active ingredient, as above defined, together withone or more acceptable carriers therefore and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand physiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and, intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the invention suitable for oral administration areprepared as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active ingredient; as a powderor granules; as solution or a suspension in an aqueous liquid or anon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredient.

For infections of the eye or other external tissues e.g. mouth and skin,the formulations are preferably applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulphoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween™ 60, Span™ 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of influenza A or B infections.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered orally, parenterally or byany other desired route. Compounds of the invention are used to providecontrolled release pharmaceutical formulations containing as activeingredient one or more compounds of the invention (“controlled releaseformulations”) in which the release of the active ingredient arecontrolled and regulated to allow less frequency dosing or to improvethe pharmacokinetic or toxicity profile of a given active ingredient.Effective dose of active ingredient depends at least on the nature ofthe condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses) or against an active influenzainfection, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. It can be expected to be from about 0.0001 to about100 mg/kg body weight per day. Typically, from about 0.01 to about 10mg/kg body weight per day. More typically, from about 0.01 to about 5mg/kg body weight per day. More typically, from about 0.05 to about 0.5mg/kg body weight per day. For example, for inhalation the dailycandidate dose for an adult human of approximately 70 kg body weightwill range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, andmay take the form of single or multiple doses.

In one implementation, active ingredients of the invention are also usedin combination with other active ingredients. Such combinations areselected based on the condition to be treated, cross-reactivities ofingredients and pharmaco-properties of the combination. For example,when treating viral infections of the respiratory system, in particularinfluenza infection, the compositions of the invention are combined withantivirals (such as amantidine, rimantadine and ribavirin), mucolytics,expectorants, bronchialdilators, antibiotics, antipyretics, oranalgesics. Ordinarily, antibiotics, antipyretics, and analgesics areadministered together with the compounds of this invention.

Another implementation of the invention includes in vivo metabolicproducts of the compounds described herein, to the extent such productsare novel and unobvious over the prior art. Such products may result forexample from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes.

Accordingly, the invention includes novel and unobvious compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof. Such products typically are identified by preparing aradiolabeled (e.g. ¹⁴C or ³H) compound of the invention, administeringit parenterally in a detectable dose (e.g. greater than about 0.5 mg/kg)to an animal such as rat, mouse, guinea pig, monkey, or to man, allowingsufficient time for metabolism to occur (typically about 30 seconds to30 hours) and isolating its conversion products from the urine, blood orother biological samples. These products are easily isolated since theyare labeled (others are isolated by the use of antibodies capable ofbinding epitopes surviving in the metabolite). The metabolite structuresare determined in conventional fashion, e.g. by MS or NMR analysis. Ingeneral, analysis of metabolites is done in the same way as conventionaldrug metabolism studies well-known to those skilled in the art. Theconversion products, so long as they are not otherwise found in vivo,are useful in diagnostic assays for therapeutic dosing of the compoundsof the invention even if they possess no neuraminidase inhibitoryactivity of their own.

Prodrugs of the novel phosphonate congeners are contemplated. Both thepolar phosphonate and guanidinium groups may be optionally furtherfunctionalized by techniques known in the art to enhance pharmacokineticand/or pharmacodynamic properties. For example, formulation and use ofprodrugs, e.g. acyloxymethyl- and aryl phosphonate esters, may beutilized to enhance oral bioavailability (Krise and Stella, Adv. DrugDeliv. Rev. 1996, 19, 287).

In one aspect of the invention, samples suspected of containingneuraminidase include natural or man-made materials such as livingorganisms; tissue or cell cultures; biological samples such asbiological material samples (blood, serum, urine, cerebrospinal fluid,tears, sputum, saliva, tissue samples, and the like); laboratorysamples; food, water, or air samples; bioproduct samples such asextracts of cells, particularly recombinant cells synthesizing a desiredglycoprotein; and the like. Typically the sample will be suspected ofcontaining an organism which produces neuraminidase, frequently apathogenic organism such as a virus. Samples can be contained in anymedium including water and organic solvent/water mixtures. Samplesinclude living organisms such as humans, and man-made materials such ascell cultures.

The treating step of the invention comprises adding the composition ofthe invention to the sample or it comprises adding a precursor of thecomposition to the sample. The addition step comprises any method ofadministration as described above. If desired, the activity ofneuraminidase after application of the composition can be observed byany method including direct and indirect methods of detectingneuraminidase activity. Quantitative, qualitative, and semiquantitativemethods of determining neuraminidase activity are all contemplated.Typically one of the screening methods described above are applied,however, any other method such as observation of the physiologicalproperties of a living organism are also applicable.

Organisms that contain neuraminidase include bacteria (Vibrio cholerae,Clostridium perfringens, Streptococcus pneumoniae, and Arthrobactersialophilus) and viruses (especially orthomyxoviruses or paramyxovirusessuch as influenza virus A (e.g. H1N1, H5N1), and B, parainfluenza virus,mumps virus, Newcastle disease virus, fowl plague virus, and sendaivirus). Inhibition of neuraminidase activity obtained from or foundwithin any of these organisms is within the objects of this invention.The virology of influenza viruses is described in “Fundamental Virology”(Raven Press, New York, 1986), Chapter 24. The compounds of thisinvention are useful in the prophylaxis of influenza infections ortreatment of existing influenza infections in animals such as ducks andother birds, rodents, swine, or in humans.

Compositions of the invention are screened for inhibitory activityagainst neuraminidase by any of the conventional techniques forevaluating enzyme activity. Within the context of the invention,typically compositions are first screened for inhibition ofneuraminidase in vitro and compositions showing inhibitory activity arethen screened for activity in vivo. Compositions having in vitro K_(i)(inhibitory constants) of less than about 5×10⁻⁶ M, typically less thanabout 1×10⁻⁷ M and preferably less than about 5×10⁻⁸ M are preferred forin vivo use.

Useful in vitro screens have been described in detail and will not beelaborated here. (Itzstein, M. von et al.; “Nature”, 363(6428):418-423(1993); Potier, M.; et al.; “Analyt. Biochem.”, 94:287-296 (1979);Chong, A. K. J.; et al.; “Biochem. Biophys. Acta”, 1077:65-71 (1991);and Colman, P. M.; et al.; International Publication No. WO 92/06691(Int. App. No. PCT/AU90/00501, publication date Apr. 30, 1992)). In vivoscreens have also been described in detail (Itzstein, et al., 1993 inparticular page 421, column 2, first full paragraph, to page 423, column2, first partial paragraph, and Colman, p. 36).

Table 1 shows neuraminidase inhibition, anti-influenza, and cytotoxicityactivities of zanamivir phosphonate derivatives 1a and 1b in comparisonwith zanamivir and oseltamivir acid. The phosphonate derivatives 1a and1b showed greater potencies than zanamivir against various wild-type andmutant influenza viruses.

TABLE 1 Neuraminidase inhibition, anti-influenza activity, andcytotoxicity assay Oseltamivir Bioassay* Measurement 1a 1b Zanamiviracid WSN(H1N1) IC₅₀ (nM)^(b) 0.65 ± 0.05 1.2 ± 0.4  5.3 ± 2.1  2.6 ± 1.1EC₅₀ (nM)^(c) 1.3 2.4 ± 0.8 23.5 ± 8.5 12.2 ± 2.3 WSN_274Y(H1N1) IC₅₀(nM)^(b) 0.5 0.25 ± 0.05  1.75 ± 0.75 593 ± 68 EC₅₀ (nM)^(c) 27 26 ± 8 290 ± 15 30000 ± 1600 Pandemic (H1N1) IC₅₀ (nM)^(b) 0.9 0.8 4.3 1.7 EC₅₀(nM)^(c) 20.3 26.5 267 76 RG14 (H5N1) IC₅₀ (nM)^(b) 1.0 0.8 4.0 0.6 EC₅₀(nM)^(c) 978 1700 ± 500  16360 ± 2980 1250 ± 440 Udorn (H3N2) IC₅₀(nM)^(b) 6.4 5.2 37.9 3.2 EC₅₀ (nM)^(c) 55 32 ± 23 41 ± 6  3.0 ± 1.8293T cell CC₅₀ (nM)^(d) >30,000 >50,000 >100,000 >100,000 ^(a)Influenzaviruses A/WSN/1933 (H1N1), H274Y neuraminidase mutant from A/WSN/1933(H1N1). A/California/7/2009 (pandemic H1N1), A/Vietnam/1194/2004 RG14(H5N1), and A/Udorn/307/1972 (H3N2) were used as bioassay materials forneuraminidase inhibition and anti-influenza assays. Human 293T cellswere used for cytotoxicity measurements for compounds. ^(b)A fluorescentsubstrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid(MUNANA) was used to determine the IC₅₀ values that are compoundconcentrations causing 50% inhibition of different influenzaneuraminidase enzymes. ^(c)Inhibition constants were determined bykinetic studies using MUNANA as the substrate. ^(d)The anti-influenzaactivities against different influenza strain were measured as EC₅₀values that are the compound concentrations for 50% protection of thecytopathic effects due to the infection by different influenza strains.^(e)The highest concentration used without noticeable toxic effects inthe assay of cytotoxicity on 293T cells.

The neuraminidase inhibitory assays were measured against theneuraminidase of several influenza strains using the influenzaassociated neuraminidase as the enzyme sources. A fluorescence substrateMUNANA (2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) was usedto measure the neuraminidase activities for all viral enzymes. Table 1shows the IC₅₀ values that measure the compound concentrations for 50%inhibition of neuraminidase activities and assess the relativeneuraminidase inhibitory potencies of these compounds.

The IC₅₀ values for both 1a and 1b against all five neuraminidases areall noticeably more potent than zanamivir and similar to those foroseltamivir acid. These two phosphonate compounds are significantly moreactive in inhibiting the oseltamivir-resistant neuraminidase mutant ofWSN 274Y that has a tyrosine residue replacing the parental histidine atthe 274 position of the neuraminidase.

The anti-influenza activities of these compounds were measured againstfive influenza strains for abilities to protect the influenza infectionmediated cytopathic effects. The anti-influenza activities weredetermined as EC₅₀ values that are the concentrations for 50% protectionof the infection mediated cytopathic effects. Table 1 shows that 1a and1b have greater anti-influenza activities against the H1N1 influenzaviruses, such as the WSN and the 2009 pandemic H1N1 strains.

The anti-influenza activities of 1a and 1b are particularly noticeableagainst the oseltamivir-resistant WSN(H1N1) virus. H1N1 influenzaviruses carrying the oseltamivir-resistant neuraminidase mutation at the274 position have been the prevailing H1N1 isolates in clinic (Moscona,A. N. Engl. J. Med. 2005, 353, 2633). The superior anti-influenzaactivities of 1a and 1b for this mutant neuraminidase may impact theoption for treating this prevailing influenza strain. In addition tobeing potent anti-influenza inhibitors against the H1N1 influenzastrains, 1a and 1b are also comparable to zanamivir as anti-influenzaagents against the RG14 (H5N1) and the Udorn (H3N2) influenza strains.

Bioassay measurements showed that both 1a and 1b are potentanti-neuraminidase and anti-influenza compounds. They are generally morepotent than zanamivir and are very active against theoseltamivir-resistant H1N1 influenza viruses. In addition to be potentanti-influenza agents, they are nontoxic to the human 293T cells at thehighest testing concentrations (Table 1).

In one implementation of the present disclosure, phosphonate 1a is apotent NA inhibitor and anti-flu agent against A/WSN/1933 (H1N1) viruswith IC₅₀ and EC₅₀ values of 0.65 and 1.3 nM, respectively.

In one implementation of the present disclosure, phosphonate 1a is apotent NA inhibitor and anti-flu agent against the H274Y mutant ofA/WSN/1933 (H1N1) virus with IC₅₀ and EC₅₀ values of 0.5 and 27 nM,respectively. These IC₅₀ and EC₅₀ values of the zanamivir phosphonateare particularly impressive for Tamiflu-resistant H1N1 virus.

In one implementation of the present disclosure, phosphonate 1a is alsoa potent NA inhibitor and anti-flu agent against A/California/7/2009(pandemic H1N1), A/Vietnam/1194/2004 RG14 (H5N1), and A/Udorn/307/1972(H3N2) viruses.

In one implementation of the present disclosure, phosphonate 1b is apotent NA inhibitor and anti-flu agent against A/WSN/1933 (H1N1) viruswith IC₅₀ and EC₅₀ values of 1.2 and 2.4 nM, respectively.

In one implementation of the present disclosure, phosphonate 1b is apotent NA inhibitor and anti-flu agent against the H274Y mutant ofA/WSN/1933 (H1N1) virus with IC₅₀ and EC₅₀ values of 0.25 and 26 nM,respectively. The high potency of 1b against Tamiflu-resistant H1N1virus is very significant.

In one implementation of the present disclosure, phosphonate 1b is alsoa potent NA inhibitor and anti-flu agent against A/California/7/2009(pandemic H1N1), A/Vietnam/1194/2004 RG14 (H5N1), and A/Udorn/307/1972(H3N2) viruses.

In one aspect of the invention, molecular modeling of theneuraminidase-phosphonate complex indicates a pertinent binding mode ofthe phosphonate with three arginine residues in the active site. Themolecular docking experiments (FIG. 2) using the known N1 crystalstructure (PDB code: 2HU4) reveal that the phosphonate inhibitor 1bbinds strongly with the tri-arginine residues of NA, in addition toother interactions exerted by the C₇-C₉ glyceryl, C₄-acetamido andC₅-guanidino groups in the binding pocket similar to theneuraminidase-zanamivir complex.

In one aspect of the invention, molecular modeling of phosphonateinhibitor 1a showed the strong interactions with the three arginineresidues of neuraminidase, in addition to other interactions exerted bythe C₇-C₉ glyceryl, C₄-acetamido and C₅-amino groups in the bindingpocket (FIG. 2).

Identification of Tamiflu-Resistant Viral Isolates

The specification herein discloses the design and practice of the methodof Resistance Assessment by Binding Competition (RABC) for developmentof effective diagnostics for Tamiflu-resistant viral isolates.

Zanamivir and OC bind to the same active site of influenza neuraminidase(NA). However, oseltamivir-resistance H5N1 virus neuraminidase can stillretain susceptibility to zanamivir. (Collins P J, et al. (2008). Nature453 (7199): 1258-1261). An induced fit of the neuraminidase thatinvolves the reorientation of the Glu276 residue toward Arg224 creatinga larger hydrophobic pocket is needed to accommodate the side chain ofOC (M. Z. Wang, C. Y. Tai, D. B. Mendel, Antimicrob. Agents Chemother.46, 3809 (2002); P. J. Collins et al., Nature 453, 1258 (2008)). The N1group neuraminidases can evolve to resistant forms with mutations suchas H274Y that prevent the formation of the hydrophobic pocket resultingin several hundred fold increase in the oseltamivir K, values. In theabsence of the need of a hydrophobic pocket, the zanamivir K_(i) for theH274Y neuraminidase is unchanged (P. J. Collins et al., Nature 453, 1258(2008)). In a feature of the invention, the binding difference wasexplored to develop a novel diagnosis for OC susceptibility of viralisolates.

Selection of a recognition unit (R) that binds to theoseltamivir-binding region of influenza virus neuraminidase and usingthe differential binding between oseltamivir-sensitive andoseltamivir-resistant versions of influenza virus has been surprisinglyfound to be a basis for detecting Tamiflu-resistant strains of influenzavirus.

In certain aspects of the invention the recognition unit (R) is coupledto a sensor unit (S). In some embodiments, the recognition unit (R) iscoupled to a linker (L) which is in turn coupled to a sensing unit (S).The R-L-S molecule is used to distinguish Tamiflu-resistant fromTamiflu-sensitive influenza viruses.

Being a potent neuraminidase inhibitor, oseltamivir carboxylate (OC;Tamiflu®) is an effective competitor for non-OC molecules (R) that bindto NA molecules in influenza viruses. However, OC is a poor competitorfor the binding of the OC-resistant mutants (e.g., H274Y) that stillbinds non-OC recognition units (R) equally as well as wild type NA.

The recognition unit (R) is typically selected from a known NA bindingmoiety such a zanamivir, tamiphosphor guanidine monoester, or zanamivirphosphonate 1a or its derivatives 1b, 1c and 1d. In some embodiments, Ris selected from oseltamivir-containing phosphonate compounds that haveactivity as neuraminidase inhibitors against wild-type and H274Y mutantof H1N1 and H5N1 viruses as disclosed in U.S. Pat. No. 7,888,337B2.

Binding to NA is detected by the signal from the sensing moiety (i.e.detectable moiety) can be observed. Such homogenous fluorescent andcalorimetric sensing moieties are known to those skilled in the art.See, for example: Wang Q. M. et al., “A continuous calorimetric assayfor rhinovirus-14 3C protease using peptide p-nitroanilides assubstrates” Anal. Biochem. Vol. 252, pp. 238-45 (1997), and Basak S. etal. “In vitro elucidation of substrate specificity and bioassay ofproprotein convertase 4 using intramolecularly quenched fluorogenicpeptides” Biochem. J. Vol. 380, pp. 505-14 (2004).

The sensor unit is linked directly or indirectly (viaavidin/streptavidin-biotin, antibody-antigen, or other methods known inthe art) to a detectable moiety selected from the group consisting of afluorescent label, a gold label and an enzyme label. The various kindsof sensors are not particularly limited and can be appropriatelyselected according to the purpose. Examples thereof include radioactivelabels, quantum dot labels, protein labels, and the like.

In some aspects the sensing unit forms a detectable binding complex witha conjugated binding partner, forming a binding pair, wherein saidbinding partner is conjugated to a reagent. The binding pair may bebased on any of: the binding pair is any one selected from the groupconsisting of streptavidin:biotin; avidin:biotin; folic acid:folatebinding protein; sialic acid, carbohydrates, or glycoproteins:lectins;oligo- or poly-dA:oligo- or poly-dT; oligo- or poly-dC:oligo- orpoly-dG; phenylboronic acid:salicylhydroxamic acid; aldehyde and ketonemoieties:hydrazides; sulfhydryl moiety:maleimides; aminomoieties:N-hydroxysuccinimide esters; and heavy metals:thiols; the Fcportion of IgG: Protein A/Protein G/Protein A/G; digoxigenin:anti-digoxigenin; 5-bromodeoxyuridine: anti-bromodeoxyuridine;dinitrophenyl:anti-dinitrophenyl; fluoresceinisothiocyanate:anti-fluorescein isothiocyanate;N-2-acetylaminofluorene:anti-N-2-acetylaminofluorene; andN-2-acetylamino-7-iodofluorene:anti-N-2-acetylamino-7-iodofluorene.

In certain aspects the label comprises a fluorphore. In some aspects,the fluorophore is selected from the group consisting of fluorescein,rhodamine, coumarin, resorufin, xanthene, cyanine, pyrene,phthalocyanine, phycobiliprotein, Alexa, Cy3, Cy5, squarene dye,combinations resulting in energy transfer dyes, and derivatives thereof.The Alexa fluorescent dye may be selected from the group consisting ofAlexa Fluor 647, Alexa Fluor 546 and Alexa Fluor 532. In someembodiments the fluorophore is selected from the group consisting of:BODIPY maleimides, iodoacetamides and methyl bromides; Alexa Fluormaleimides; fluorescein 5- and 6-isomer maleimides and methyl bromides;Oregon Green isothiocyanates and maleimides; tetramethylrhodamine 5- and6-isomer iodoacetamides and maleimides; Rhodamine Red maleimides; TexasRed bromoacetamides and maleimides; pyridyloxazole maleimides;benzoxadiazole derivatives including NBD halides and iodoacetamides,SBD; Lucifer Yellow iodoacetamide; stilbene iodoacetamides andmaleimides; coumarin maleimides and iodoacetamides, i.e. MDCC, IDCC, andothers; naphthalene derivatives, i.e. acrylodan, badan, IAANS, MIANS,IAEDANS, and Dansyl; aziridine; dapoxyl derivatives, i.e. dapoxyl(2-bromoacetamidly)sulfonamide; pyrene maleimides and iodoacetylderivatives; and monobromo- and monochlorobimanes.

The method or means for detecting the signal from the sensor unit (S) isnot particularly limited and can be appropriately selected according tothe purpose. For example, when the signal is emission, quenching, etc.,it is detected by a photodetector, camera, etc.

The combination of the light-emitting portion and the quenching portionis not particularly limited and can be appropriately selected accordingto the purpose. For example, those known as the technique offluorescence resonance energy transfer (FRET), etc. can be suitablyadopted.

The light-emitting portion is not particularly limited as long as it cangenerate emission, and can be appropriately selected according to thepurpose. Examples thereof include those containing a fluorescentsubstance, chemiluminescent substance, electrochemiluminescentsubstance, etc. or those formed of these substances. These may be usedalone or two or more may be used in combination. Among these, in thecase where the quenching portion is present adjacent to thelight-emitting portion, those of which emission is quenched by theaction of the quenching portion are preferable, the fluorescentsubstance is more preferable in that visibility thereof is excellent anddetection is easy. The fluorescent substance is not particularly limitedand can be appropriately selected according to the purpose. Examplesthereof include anthracene, fluorescein, fluorescein isothiocyanate(FITC), rhodamines such as tetramethyl rhodamine and sulforhodamine,dansyl chloride, Texas Red, AL 350, indocarbocyanine (CY), and the like.

The quenching portion is not particularly limited if it can quench theemission of the light-emitting portion when the quenching portion islocated adjacent to the light-emitting portion, and the quenchingportion can be appropriately selected according to the type, etc. of thelight-emitting portion. Examples thereof include those containing aquenching substance, or those formed of the quenching substance. Thequenching substance is not particularly limited and can be appropriatelyselected according to the purpose. When the light-emitting portion isformed of the fluorescent substance, examples of the quenching substanceinclude substances capable of absorbing the energy released when thefluorescent substance emits light, and the like. Suitable examplesinclude substances allowing fluorescence resonance energy transfer(FRET) between the light-emitting substance. Specific examples includetetramethylrhodamine isothiocyanate (TRITC),dimethylaminobenzenesulfonyl (DABSYL), gold nanoparticles, Black HoleQuencher, and the like.

The recognition unit is conjugated to a sensor which is in turn coupledto a light-emitting portion and a quenching portion. The quenchingportion can quench emission of the light-emitting portion before therecognition unit binds to the target NA, i.e., when the quenchingportion is present adjacent to the light-emitting portion. After therecognition unit has bound to the target NA, the quenching portiondissociates from the target nucleic acid. As a result, the quenchingportion exists away from the light-emitting portion, resulting in theloss of the action of the quenching portion and allowing thelight-emitting portion to generate emission.

The linker unit (L) is not particularly limited and can be appropriatelyselected as long as it does not impair the effect of the invention. Forexample, a water-soluble linker such as ethylene glycol linker can beused. The length of the water-soluble linker can be determined to anappropriate length.

In one aspect, either the influenza virus sample to be tested or theR-L-S complex is immobilized on a solid surface as an array. The solidsurface may be selected from the group consisting of Langmuir Blodgettfilm, glass, germanium, silicon, (poly)tetrafluoroethylene, polystyrene,gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, andcombinations thereof. The solid surface may be any one selected from thegroup consisting of a microwell or microtiter plates or dishes, siliconchips, glass slides, beads, microparticles, films or membranes, bottles,dishes, slides, blotting material, filters, fibers, woven fibers, shapedpolymers, particles, dip-sticks, test tubes, chips, and microchips.

In one embodiment, the influenza virus and/or virus particles are boundto a support containing at least one type of carbohydrate receptorselected from the group consisting of natural or syntheticoligosaccharide, which is conjugated to, or situated in composition withglycoproteins like glycophorin, α1-acid glycoprotein, α2-macroglobulin,ovomucoid, and combinations thereof which carbohydrate receptor binds tothe hemagglutinin component of the viruses and/or virus particles. Inparticular, influenza viruses and/or virus particles comprising allknown Avian Influenza (AI) sub-types that are normally sensitive toTamiflu® can detected. The method is suitable to detect influenzaviruses and/or virus particles comprising a highly pathogenic variantthat is resistant to Tamiflu®. The invention can be performed inparticular with a support which is a chromatographic paper or membrane.Such materials are well-known to the skilled person. According to theinvention it is possible to covalently attach or physically adsorb thecarbohydrate receptor to the support. (US Pub. Pat App. Ser. No.20100009339).

Methods of the invention can be used with suspected influenza viruscontaining samples selected from the group consisting of mucus, saliva,respiratory secretion, throat wash, nasal wash, spinal fluid, sputum,urine, semen, sweat, feces, plasma, blood, broncheoalveolar fluid,vaginal fluid, tear fluid and tissue biopsy. Similar techniques are alsoapplicable for use with virus samples from cell culture, e.g., influenzasamples grown in cells such as Vero cells, etc. The methods of theinvention are suitable for practicing on intact influenza virus-infectedcells or cell-free samples of influenza virus. The influenza virus canbe of mammalian (human, equine, swine, etc.) or avian origin.

Kits

Kits which comprise the neuraminidase binding molecules of the presentinvention are also envisaged. The different kit components may bepackaged in separate containers and admixed immediately before use. Suchpackaging of the components separately may permit long-term storagewithout losing the active components' functions. Embodiments in whichtwo or more of components are found in the same container are alsocontemplated. An exemplary kit may comprise one or more of the followingreagents: a wash buffer reagent for use using heterogeneous assays; anegative control reagent free of a neuraminidase-binding capability; asignal generation reagent for development of a detectable signal fromthe signaling moiety; and a sample collection means such as a syringe,throat swab, or other sample collection device. The kits of the presentinvention may, if desired, be presented in a pack which may contain oneor more units of the kit of the present invention. The pack may beaccompanied by instructions for using the kit. The pack may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of laboratory supplements, which notice is reflective of approvalby the agency of the form of the compositions.

Exemplary Embodiments of the Binding Molecules

The invention relates to detection of Tamiflu-resistant influenza virusstrains by determining the binding ability of specific binding moleculeshaving the structure recognition unit (R)-optional linker (L)-sensingunit (S).

Examples of some embodiments of the binding molecule (BM) according tothe invention are:

BM 1: Zanamivir-Triazole Linker-Biotin

The linker containing a triazole group is formed by click chemistry(1,3-dipolar cycloaddition of alkyne and azide). Fluorescence labeledstreptavidin is used to detect the biotin unit.

BM 2: Zanamivir-Triazole Linker-Fluorescein

Fluorescein has an absorption maximum at 494 nm and emission maximum of521 nm (in water). Fluorescein isothiocyanate (FITC) is used to connectwith the linker. Alternatively, other fluorescent entities such as Alexadyes and quantum dots, can be used.

BM 3: Zanamivir-Ethylene Glycol Linker-Biotin

The length of ethylene glycol linker can be tuned from x=1 to x=4.

BM 4: Zanamivir-Ethylene Glycol Linker-Fluorescein

BM 5: Tamiphosphor Guanidine-Linker-Biotin

Tamiphosphor guanidine binds both Tamiflu-sensitive andTamiflu-resistant strains (H274Y) of influenza viruses. The Tamiphosphorguanidine monoester can be in the salt form, such as the ammonium salt.The aliphatic chain can have 1-6 carbons (x=1-4) in the linker.Alternatively, the aliphatic chain can be replaced by ethylene glycolchain as shown in Example 3.

BM 6: Tamiphosphor Guanidine-Ethylene Glycol Linker-Fluorescein

BM 7: PhosphaZanamivir-Triazole Linker-Biotin

PhosphaZanamivir can be in the salt form, such as the ammonium salt.

BM 8: PhosphaZanamivir-Ethylene Glycol Linker-Fluorescein

PhosphaZanamivir can be in the salt form, such as the ammonium salt.

BM 9: PhosphaZanamivir Monoester-Ethylene Glycol Linker-Biotin

The length of ethylene glycol linker can be tuned from x=1 to x=4.

BM 10: PhosphaZanamivir Monoester-Ethylene Glycol Linker-Fluorescein

The length of ethylene glycol linker can be from x=1 to x=4.

Other exemplars of influenza NA-binding molecules of the generalstructure R-L-S are disclosed in Kale, R. R. et al., Am. Chem. Soc.2008, 130, 8169-8171; Mckimm-Breschkin, J. L. et al., Angew. Chem. Int.Ed. 2003, 42, 3118-3121; Lu, C.-P. et al., Angew. Chem. Int. Ed. 2005,44, 6888-6892; Kimura, Y. et al., Tetrahedron Lett. 2009, 50, 3205-3208.

The present disclosure for the first time reveals that NA-bindingabilities of molecules of the structure R-L-S can be used to detectTamiflu-resistant versions of influenza virus.

Competitive Binding of NA with Oseltamivir Carboxylate (OC) and R-L-SType Binding Molecules

A biotin-conjugated zanamivir (ZB) that binds influenza neuraminidaseseffectively was prepared. In the presence of oseltamivir carboxylate(OC) as the competitor for zanamivir binding, OC susceptibility could bedetermined. The OC binding competition assay confirmed the dramaticincrease of OC resistant H1N1 isolates from 2008 and the appearance ofOC resistant pandemic 2009 H1N1 in Taiwan. The Resistance Assessment byBinding Competition (RABC) assay was used to develop a prototype“point-of-care” test for OC susceptibility assessment. The RABC-assayprinciple could be generally applicable to high throughput survey andquick diagnosis for drug susceptibility of pathogens.

A biotin conjugated zanamivir (ZB) was prepared for this study (FIG. 1A,Examples). ZB was made by conjugation of a biotin through a linker tozanamivir at the 7-OH position that can be derivatized without muchreduction in neuraminidase inhibition (D. M. Andrews et al., Eur. J.Med. Chem. 34, 563 (1999); T. Honda et al., Bioorg. Med. Chem. Lett. 12,1925 (2002); W. H. Wen et al., J. Med. Chem. 52, 4903 (2009)). The IC₅₀value of ZB against neuraminidase was found to be 7.7 nM, higher thanthat of zanamivir (2.1 nM). ZB binding to influenza neuraminidase wasdemonstrated using neuraminidase transfected cells (FIG. 4), influenzainfected cells (see Examples), and immobilized influenza viruses (FIG.1B). Being a potent neuraminidase inhibitor, oseltamivir carboxylate(OC) could be an effective competitor for ZB binding to influenzaviruses.

Nevertheless, OC was also expected to be a poor competitor for thebinding of the OC-resistant H274Y mutant that ZB binds equally well.FIG. 1B shows that significant OC inhibition (p<0.001) on ZB binding wasshown in the measurements of 274H influenza virus with titers in therange of 10³-10⁵ PFU. A residual ZB binding about 3% was noticed in OCcompetition of higher titer 274H viral samples. In contrast, significantinhibition of ZB binding by competing OC was not observed in the OCresistant 274Y WSN virus at the same titers.

The same observations were demonstrated using transformed and influenzainfected cells (FIGS. 11-14). We named this assay Resistance Assessmentby Binding Competition, or RABC in short. We also showed that the RABCassay allows the detection of the OC-resistant 274Y mutants in mixedpopulations at greater than 10% resistant contents (FIG. 14).

A total of 137 Taiwan seasonal H1N1 clinical isolates collected in theyears 2005-2009 were queried by the RABC assay for OC susceptibilities.The viral samples were immobilized in anti-H1N1 coated microplate wellsand tested for binding using either 30 nM ZB or 30 nM ZB plus 150 nM OCin duplicate assays. The 5% residual ZB binding was used as the cut-offfor OC susceptibility determination. The RABC assay results suggestedthat all tested seasonal H1N1 isolates collected before 2008 (FIG. 8A)or early in 2008 (FIG. 8B) were OC susceptible. Not until mid 2008,OC-resistant H1N1 was isolated in Taiwan (FIG. 8B). Similarly, 48 out of50 seasonal H1N1 collected in 2009 were scored as OC resistant (FIG.8C). We also examined the OC susceptibility of the pandemic 2009 H1N1isolates collected in Taiwan. FIG. 8D shows that isolates ##1-4 andisolates ##5-7 are OC susceptible and OC resistant viruses,respectively. The RABC assays of the seven strains were conducted inblind, and the results were later shown to be consistent with their OCsusceptibility and sequence results. To evaluate the susceptibilityprediction using the RABC assay, 60 isolates of the seasonal H1N1samples described in FIG. 8A-C were randomly picked to analyze theirneuraminidase sequences. All samples predicted as OC sensitive by theRABC assay have histidines, while those predicted to be OC resistanthave tyrosines at the corresponding residue 274 of the neuraminidases(Table 2). To seek the possible presence of evolving resistantpopulations, we took a closer look of those samples with 3-5% residualZB binding values. Analyses of several isolated viral plaques from thesesamples failed to identify OC resistant viruses by binding or sequenceanalyses.

Table 2 lists all Taiwan clinical H1N1 isolates used in this study fordetermination of their OC susceptibility status by the RABC assays.Sixty viral isolates were randomly selected to determine their sequencesat amino acid 274 (AA₂₇₄; N2 nomenclature) of the viral neuraminidase.In addition, a few samples were tested using the quick assay on membranefor OC susceptibility determinations. No discrepancies were found in allassay results, indicating that the RABC is reliable for OCsusceptibility determinations.

TABLE 2 OC susceptibility determination of Taiwan seasonal H1N1isolates. The OC susceptibility of each influenza isolate determined bythe RABC assay is designated as R (resistant) or S (susceptible)according to the results of FIG. 8. Several isolates were randomlypicked for confirmation by analysis of AA₂₇₄ and (or) quick test on amembrane (the numbers in parentheses corresponding to the sample numberused in FIG. 8B). Name* OC^(b) AA₂₇₄ Quick test 2009-03510 R Tyr2009-02548 R Tyr 2009-02020 R 2009-01051 R 2009-01022 R 2009-00516 R2009-00513 R 2009-00512 R 2009-01524 R Tyr 2009-00521 R 2009-00519 R2009-00518 R 2009-04512 R 2009-00511 R 2009-03518 R 2009-03019 R Tyr2009-03005 R Tyr 2009-90010 R 2009-02530 R Tyr 2009-02027 R Tyr2009-02031 R 2009-01026 R Tyr 2009-04511 R Tyr 2009-01518 R 2009-01516 R2009-04017 R 2009-03522 R Tyr 2009-03507 R 2009-03003 R Tyr 2009-03002 RTyr 2009-02022 R 2009-01043 R 2009-03006 R 2009-00024 R Tyr 2009-04010 RTyr 2009-00515 R Tyr 2009-00514 R 2009-00510 R 2009-04503 R Tyr2009-01514 R 2009-01021 s His 2009-02013 R 2009-04007 R 2009-02010 R Tyr2009-01522 s His 2008-03020 s His S(73) 2009-00009 R 2009-00008 R Tyr2008-11526 R Tyr 2008-00846 R 2008-00842 R 2008-09219 R 2008-00843 R2008-02904 R 2008-09202 R 2008-05878 R Tyr 2008-09201 R 2008-06020 s HisS(64) 2008-05879 R Tyr 2008-02906 R 2008-08884 s His 2008-09200 R Tyr2008-09199 R 2008-02415 R Tyr 2008-00841 R 2008-10128 R Tyr 2008-02418 R2008-05877 R Tyr 2008-00302 R 2008-09198 R 2008-90003 s S(49) 2008-09020s His 2008-11548 R 2008-05866 R Tyr 2008-05860 R Tyr 2008-05859 R Tyr2008-05858 R Tyr 2008-05857 R Tyr 2008-11547 R 2008-00286 R Tyr2008-05855 R Tyr 2008-02901 R 2008-00279 R Tyr 2008-00839 R 2008-00275 R2008-08451 R Tyr 2008-00273 R 2008-09159 R Tyr 2008-09042 R Tyr R(33)2008-08949 R Tyr 2008-08885 s His 2008-05853 R Tyr 2008-08416 s His2008-00258 s His 2008-10103 R Tyr R(26) 2008-06758 R Tyr 2008-02885 RTyr 2008-05723 s His 2008-10099 R Tyr 2008-10095 s His 2008-08319 s His2008-02832 R Tyr R(19) 2008-00233 s His S(18) 2008-05506 s His S(17)2008-07903 s 2008-07895 R R(16) 2008-07860 R Tyr R(13) 2008-02808 s His2008-05815 s His 2008-03293 s 2008-09169 s 2008-04240 s 2008-03135 s2008-02612 s 2008-04226 s 2008-04063 s 2008-04169 s 2007-05601 s2007-02774 s 2007-02578 s 2007-05222 s 2007-04656 s 2007-02820 s2007-02523 s 2007-02864 s 2007-03700 s 2007-02782 s 2006-05288 s2006-05542 s 2006-00061 s 2006-05751 s 2006-00010 s 2006-04130 s2005-05524 s 2005-10393 s 2005-05515 s 2005-03468 s

 virus 

hoc susceptibility

indicates data missing or illegible when filed

Point of Care Assay

Disclosed herein is a prototype assay on membrane for visual assessmentof the OC susceptibility state of the influenza viruses. FIG. 9A showsthat the staining of the OC susceptible H1N1 (WSN) by ZB was blocked byOC competition while the staining of the OC resistant 274Y WSN wasresistant to the same competition. We used the staining on membrane toconfirm the status of the OC susceptibility of several seasonal andpandemic H1N1 isolates (FIGS. 9B and 9C). The sensitive ZB binding wasalso demonstrated on H1N1, H3N2, H5N1, and flu B influenza viruses thatwere directly immobilized on a membrane (FIG. 9D). The prototype RABCbased staining assay is quick and requires no instruments fordetections. It could be used as a “point-of-care” assay in a doctor'soffice for timely decision on treatment options for the seasonal H1N1 orpandemic H1N1 infections. In addition, the test could be used todifferentiate influenza virus versus other respiratory infections asmost other respiratory infective pathogens do not have neuraminidases.

The RABC assay is a simple and robust method and has been demonstratedon different influenza viruses and infected cells using a variety ofassay platforms. The assay principle could be applicable to otherpathogen targets for drug susceptibility assessments.

EXAMPLES

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory or scheme of action.

All the reagents were commercially available and used without furtherpurification unless indicated otherwise. All solvents were anhydrousgrade unless indicated otherwise. All non-aqueous reactions were carriedout in oven-dried glassware under a slight positive pressure of argonunless otherwise noted. Reactions were magnetically stirred andmonitored by thin-layer chromatography on silica gel. Flashchromatography was performed on silica gel of 60-200 m particle size.Yields are reported for spectroscopically pure compounds. Melting pointswere recorded on an Electrothermal MEL-TEMP® 1101D melting pointapparatus and are not corrected. ¹H and ¹³C NMR spectra were recorded onBruker AVANCE 600 spectrometer. The ³¹P NMR spectra were recorded onBruker AVANCE 500 spectrometer. Chemical shifts are given in δ valuesrelative to tetramethylsilane (TMS); coupling constants J are given inHz. Internal standards were CDCl₃ (δ_(H)=7.24), MeOH-d₄ (δ_(H)=3.31) orD_(2O) (δ_(H)=4.79) for ¹H-NMR spectra, CDCl₃ (δ_(c)=77.0) or MeOH-d₄(δ_(c)=49.15) for ¹³C-NMR spectra, and H₃PO₄ in D_(2O) (δ_(P)=0.00) for³¹P-NMR spectra. The splitting patterns are reported as s (singlet), d(doublet), t (triplet), q (quartet), m (multiplet), br (broad) and dd(double of doublets). IR spectra were recorded on a Thermo Nicolet 380FT-IR spectrometer. Optical rotations were recorded on a Perkin-ElmerModel 341 polarimeter. High resolution ESI mass spectra were recorded ona Bruker Daltonics spectrometer.

Example 13-Acetamido-4,6-diacetoxy-2-(1,2,3-triacetoxy)propyl-3,4,5,6-tetrahydro-2H-pyran(3)

Under an atmosphere of nitrogen, a suspension of N-acetylneuraminic acid(5 g, 16.2 mmol) in pyridine (75 mL) and acetic anhydride (75 mL) wasstirred at room temperature for 12 h, and then heated at 100° C. for 5h. The reaction mixture was cooled to room temperature, and concentratedunder reduced pressure. The residual brownish glassy oil was dissolvedin CH₂Cl₂ (150 mL), and washed successively with saturated aqueousNaHCO₃ (100 mL), aqueous 1 M HCl (100 mL) and brine (100 mL). Theorganic layer was dried over MgSO₄, filtered and concentrated. Thebrownish residue was purified by column chromatography on silica gel(EtOAc/hexane, 67:33 to 100:0) to afford 3 as a pale yellow foam (3.8 g,50%), which contained inseparable mixture of anomers (α/β=1:5). Theanomeric mixture of 3 was used in the next step without furtherseparation. C₂₀H₂₉NO₁₂; TLC (EtOAc) R_(f)=0.35; ¹H NMR (600 MHz, CDCl₃)δ 6.26 (0.83H, d, J=2.5 Hz, H-1β), 5.62 (0.17H, dd, J=10.3, 2.1 Hz,H-1α), 5.43 (0.17H, ddd, J=6.1, 4.4, 1.9 Hz), 5.29-5.27 (1.66H, m), 5.22(0.83H, td, J=10.6, 4.9 Hz), 5.17 (0.83H, td, J=6.5, 2.7 Hz), 5.11-5.07(0.34H, m), 5.03 (0.17H, ddd, J=6.5, 2.7 Hz), 4.36 (0.17H, dd, J=12.5,2.6 Hz), 4.31 (0.83H, dd, J=12.5, 2.8 Hz), 4.08-3.98 (2.83H, m), 3.74(0.17H, dd, J=10.5, 2.5 Hz), 2.17-2.15 (0.17H, m), 2.15-2.13 (0.83H, m),2.11 (2.49H, s), 2.10 (0.51H, s), 2.09 (0.51H, s), 2.08 (2.49H, s), 2.07(0.51H, s), 2.04 (2.49H, s), 2.03 (0.51H, s), 2.017 (2.49H, s), 2.013(0.51H, s), 2.00 (2.49H, s), 2.00-1.98 (0.83H, m), 1.98-1.96 (0.17H, m),1.88 (2.49H, s), 1.87 (0.51H, s).

Example 2 Diethyl(5-acetamido-4-acetoxy-6-(1,2,3-triacetoxy)propyl-3,4,5,6-tetrahydro-2H-pyran-2-yl)phosphonate (4)

The anomeric mixture of 3 (2.15 g, 4.52 mmol) and diethyl trimethylsilylphosphite (3.11 mL, 13.65 mmol) in anhydrous CH₂Cl₂ (30 mL) was treatedwith trimethylsilyl trifluoromethylsulfonate (TMSOTf, 1.23 mL, 6.78mmol) at 0° C. After 30 min, the mixture was warmed to room temperature,and stirred for 24 h. The mixture was poured into ice water (20 mL), andthe aqueous layer was extracted with CH₂Cl₂ (20 mL, 2×). The combinedextracts were washed successively with saturated aqueous NaHCO₃ (50 mL)and brine (50 mL), dried over MgSO₄, filtered and concentrated. Theresidue was purified by column chromatography on silica gel(acetone/EtOAc, 1:9) to afford 4 as a colorless syrup (1.55 g, 62%),which contained a mixture of the α- and β-anomers (2:3). The anomericmixture of phosphonate 4 was used in the next step without furtherseparation. The analytical samples of pure α- and β-anomers (4a and 413)were obtained by flash column chromatography on silica gel(EtOAc/acetone, 100:0 to 90:10).

α-Anomer 4α: C₂₂H₃₆NO₁₃P; colorless foam; TLC (EtOAc/acetone, 9:1)R_(f)=0.25; (α)_(D) ²⁰=+39.4 (c=4.6, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ5.30 (1H, dd, J=5.7, 1.7 Hz), 5.24 (1H, d, J=9.9 Hz, NH), 5.18 (1H, td,J=6.6, 2.5 Hz), 4.98 (1H, td, J=10.6, 5.0 Hz), 4.40 (1H, dd, J=12.3, 5.0Hz), 4.22-4.09 (5H, m), 3.97 (1H, q, J=10.1 Hz), 3.74 (1H, td, J=12.5,2.4 Hz), 3.62 (1H, dd, J=10.3, 2.0 Hz), 2.27 (1H, dd, J=12.8, 4.9 Hz),2.09 (3H, s), 2.05 (3H, s), 2.02 (3H, s), 2.01 (3H, s), 1.98-1.92 (1H,m), 1.87 (3H, s), 1.35-1.31 (6H, m); ¹³C NMR (150 MHz, CDCl₃) δ 170.9(C), 170.5 (C), 170.3 (C), 170.2 (C), 170.1 (C), 79.0 (CH, d,3J_(c-p)=17.3 Hz), 71.8 (CH, d, ¹J_(c-p)=174.6 Hz, C-1), 71.6 (CH, d,³J_(c-p)=20.9 Hz), 71.0 (CH), 67.9 (CH), 63.4 (CH₂, d, ²J_(c-p)=6.9 Hz,POCH₂), 62.8 (CH₂, d, ²J_(c-p)=6.2 Hz, POCH₂), 62.2 (CH₂, C-8), 49.6(CH, C-4), 31.3 (CH₂, C-2), 23.1 (CH₃), 20.9 (CH₃), 20.8 (CH₃), 20.7(CH₃, 2×), 16.5 (CH₃, d, ³J_(c-p)=5.4 Hz, POCH₂CH₃), 16.3 (CH₃, d,³J_(c-p)=5.4 Hz, POCH₂CH₃); ³1P NMR (202 MHz, CDCl₃) δ 18.48; HRMS calcdfor C_(22H35NO)1_(3P): 552.1846. found: m/z 552.1921 (M−H)⁺.

β-Anomer 4β: C_(22H36NO13P); colorless foam; TLC (EtOAc/acetone, 9:1)R_(f)=0.28; (α)_(D) ²⁰=−40.1 (c=3.0, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ5.45 (1H, d, J=10.1 Hz, NH), 5.35 (1H, dd, J=7.3, 2.3 Hz), 5.32 (1H, td,J=15.0, 4.8 Hz), 5.21-5.18 (1H, m), 4.45 (1H, d, J=10.0 Hz), 4.33 (1H,dd, J=12.4, 2.8 Hz), 4.30 (1H, dd, J=12.3, 7.1 Hz), 4.19-4.13 (2H, m),4.12-4.04 (4H, m), 2.35-2.31 (1H, m), 2.11 (3H, s), 2.08 (3H, s), 2.017(3H, s), 2.011 (3H, s), 2.09-2.03 (1H, m), 1.88 (3H, s), 1.34 (3H, t,J=7.0 Hz), 1.33 (3H, t, J=7.0 Hz); ¹³C NMR (150 MHz, CDCl₃) δ 170.8 (C),170.6 (C), 170.2 (C), 170.1 (C), 169.8 (C), 74.0 (CH), 69.7 (CH), 69.5(CH), 67.9 (CH, d, ¹Jc-p=157.2 Hz, C-1), 67.7 (CH), 63.0 (CH₂, d,²J_(c-p)=7.2 Hz, POCH₂), 62.7 (CH₂, d, ²J_(c-p)=6.6 Hz, POCH₂), 62.0(CH₂, C-8), 49.0 (CH, C-4), 29.5 (CH₂, d, ²J_(c-p)=3.2 Hz, C-2), 23.1(CH₃), 21.0 (CH₃), 20.9 (CH₃), 20.7 (CH₃, 2×), 16.2 (CH₃, d,³J_(c-p)=5.1 Hz, POCH₂CH₃), 16.3 (CH₃, d, ³J_(c-p)=5.1 Hz, POCH₂CH₃);³¹P NMR (202 MHz, CDCl₃) δ 21.36; HRMS calcd for C_(22H35NO13P):552.1846. found: m/z 552.1879 (M−H)⁺.

Example 3 Diethyl(5-acetamido-4-acetoxy-6-(1,2,3-triacetoxy)propyl-4,5,6-trihydropyran-2-yl)phosphonate (5)

The anomeric mixture of phosphonate 4 (1.1 g, 2 mmol) andN-bromosuccinimide (885 mg, 5 mmol) in anhydrous CH₂Cl₂ (20 mL) washeated to reflux under irradiation from a 100 W tungsten lamp. Theprogress of reaction was monitored by TLC. On completion (.about. 6 h)the mixture was cooled to room temperature, and the precipitatesuccinimide was filtered off. The filtrate was evaporated under reducedpressure to give a crude 2-bromo derivative as yellow syrup, which wasused in the next step without further purification.

A solution of the above-prepared bromo compound in anhydrous pyridine(10 mL) was stirred at 50° C. for 2 h. The solution was concentratedunder reduced pressure, and the brown residue was purified by columnchromatography on silica gel (EtOAc/acetone, 100:0 to 90:10) to affordconjugated phosphonate 5 as colorless foam (827 mg, 75% for two steps).C₂₂H₃₄NO₁₃P; TLC (EtOAc/acetone, 9:1) R_(f)=0.28; (α)_(D) ²⁰=+43.8(c=0.59, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 5.74 (1H, dd, J=10.7, 2.2Hz), 5.54 (1H, d, J=8.2 Hz, NH), 5.42-5.40 (2H, m), 5.26 (1H, td, J=6.4,2.9 Hz), 4.39-4.34 (2H, m), 4.29 (1H, q, J=9.1 Hz), 4.17-4.09 (5H, m),2.09 (3H, s), 2.05 (3H, s), 2.04 (3H, s), 2.02 (3H, s), 1.91 (3H, s),1.35 (3H, t, J=7.0 Hz), 1.31 (3H, t, J=7.0 Hz); ¹³C NMR (150 MHz, CDCl₃)δ 170.8 (C), 170.4 (C), 170.3 (C), 169.8 (C), 169.7 (C), 147.8 (C, d,¹J_(c-p)=225 Hz, C-1), 113.0 (CH, d, ²J_(c-p)=22.8 Hz, C-2), 76.5 (CH,d, ³J_(c-p)=9.3 Hz), 69.9 (CH), 68.4 (CH, d, ³J_(c-p)=15.2 Hz), 67.2(CH), 63.2 (CH₂, d, ²J_(c-p)=5.4 Hz, POCH₂), 63.0 (CH₂, d, ²J_(c-p)=5.7Hz, POCH₂), 61.8 (CH₂, C-8), 46.4 (CH, C-4), 23.0 (CH₃), 20.78 (CH₃),20.73 (CH₃), 20.63 (CH₃), 20.60 (CH₃), 16.16 (CH₃, d, ³J_(c-p)=4.8 Hz,POCH₂CH₃), 16.12 (CH₃, d, ³J_(c-p)=4.8 Hz, POCH₂CH₃); ³¹P NMR (202 MHz,CDCl₃) δ 6.374; HRMS calcd for C₂₂H₃₃NO₁₃P: 550.1690. found: m/z550.1684 (M−H)⁺.

Example 4 Diethyl(4-(1,2,3-triacetoxy)propyl-2-methyl-3a,7a-dihydro-4H-pyrano(3,4-d)oxazol-6-yl)phosphonate(6)

To a solution of phosphonate 5 (550 mg, 1 mmol) in a mixture of aceticacid (2 mL) and acetic anhydride (2 mL) was treated with conc. H₂SO₄(0.2 mL). The mixture was stirred for 48 h at room temperature, pouredinto cold (0° C.) saturated aqueous NaHCO₃ (pH 9), and stirred for 30min before extraction with EtOAc (30 mL, 5×). The combined extracts weredried over MgSO₄, filtered and concentrated under reduced pressure. Theresidual oil was purified by column chromatography on silica gel(acetone/EtOAc, 1:9) to afford 6 as pale yellow syrup (394 mg, 80% fortwo steps). C₂₀H₃₀NO₁₁P; TLC (EtOAc/acetone, 9:1) R_(f)=0.30; (α)_(D)²⁰=−11.6 (c=0.50, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 6.20 (1H, dd,J=10.3, 4.0 Hz), 5.58 (1H, ddd, J=6.6, 2.9, 1.1 Hz), 5.38 (1H, td,J=7.7, 2.5 Hz), 4.71 (1H, ddd, J=8.6, 4.0, 2.0 Hz), 4.40 (1H, dd,J=12.4, 2.5 Hz), 4.19 (1H, dd, J=12.5, 5.9 Hz), 4.18-4.07 (4H, m), 3.93(1H, td, J=9.2, 0.6 Hz), 3.34 (1H, dd, J=10.1, 2.7 Hz), 2.11 (3H, s),2.04 (3H, s), 2.03 (3H, s), 1.98 (3H, s), 1.34 (3H, t, J=7.0 Hz), 1.32(3H, t, J=7.0 Hz); ¹³C NMR (150 MHz, CDCl₃) δ 170.5 (C), 169.7 (C),169.4 (C), 167.2 (C, N═CCH3), 150.1 (C, d, ¹J_(c-p)=225 Hz, C-1), 111.9(CH, d, ²J_(c-p)=23.4 Hz, C-2), 76.1 (CH, d, ³J_(c-p)=6.3 Hz), 71.2 (CH,d, ³J_(c-p)=15.3 Hz), 69.6 (CH), 68.8 (CH), 63.1 (CH₂, d, ²J_(c-p)=5.9Hz, POCH₂), 62.9 (CH₂, d, ²J_(c-p)=5.7 Hz, POCH₂), 61.8 (CH, C-4), 61.6(CH₂, C-8), 20.7 (CH₃), 20.6 (CH₃), 20.5 (CH₃), 16.2 (CH₃, d,³J_(c-p)=5.1 Hz, POCH₂CH₃), 16.1 (CH₃, d, ³J_(c-p)=5.1 Hz, POCH₂CH₃),14.0 (CH₃, N═CCH₃); ³¹P NMR (202 MHz, CDCl₃) δ 6.375; HRMS calcd forC₂₀H₂₉NO₁₁P: 490.1478. found: m/z 490.1374 (M−H)⁺.

Example 5 Diethyl (5-acetamido-4-azido-6-(1,2,3-triacetoxy)propyl-4,5,6-trihydropyran-2-yl) phosphonate (7)

To a solution of oxazoline 6 (393 mg, 0.8 mmol) in t-BuOH (10 mL) wastreated with azidotrimethylsilane (0.53 mL, 4 mmol) at 80° C. for 24 h.The solution was poured into saturated aqueous NaHCO₃, and extractedwith EtOAc (30 mL, 3×). The combined extracts were dried over MgSO₄,filtered and concentrated to afford the azido compound 7 as a colorlesssyrup (371 mg, 87%), which was practically pure to be used in the nextstep. An analytical sample was obtained by flash column chromatographyon silica gel (10% acetone in EtOAc). C₂₀H₃₁N_(4O11)P; TLC(EtOAc/acetone, 9:1) R_(f)=0.30; (α)_(D) ²⁰=+82.7 (c=0.58, CH₂Cl₂); ¹HNMR (600 MHz, CDCl₃) δ 5.75 (1H, dd, J=10.3, 2.4 Hz), 5.73 (1H, d, J=8.6Hz), 5.38 (1H, dt, J=7.1, 1.5 Hz), 5.26 (1H, ddd, J=8.5, 5.8, 2.6 Hz),4.53-4.50 (2H, m), 4.36 (1H, dd, J=12.5, 2.6 Hz), 4.17-4.08 (5H, m),3.67 (1H, q, J=9.2 Hz), 2.10 (3H, s), 2.05 (3H, s), 2.02 (3H, s), 1.99(3H, s), 1.34 (3H, t, J=7.1 Hz), 1.32 (3H, t, J=7.1 Hz); ¹³C NMR (150MHz, CDCl₃) δ 170.8 (C), 170.5 (C), 170.1 (C), 169.7 (C), 147.7 (C, d,¹J_(c-p)=224 Hz, C-1), 112.4 (CH, d, ²J_(c-p)=22.9 Hz, C-2), 75.9 (CH,d, ³J_(c-p)=9.2 Hz), 69.7 (CH), 67.3 (CH), 63.5 (CH₂, d, ²J_(c-p)=5.7Hz, POCH₂), 63.3 (CH₂, d, ²J_(c-p)=5.9 Hz, POCH₂), 61.9 (CH₂, C-8), 57.8(CH, d, ³J_(c-p)=14.7 Hz), 48.5 (CH, C-4), 23.2 (CH₃), 20.8 (CH₃), 20.77(CH₃), 20.71 (CH₃), 16.27 (CH₃, d, ³J_(c-p)=5.7 Hz, POCH₂CH₃), 16.23(CH₃, d, ³J_(c-p)=5.7 Hz, POCH₂CH₃).

Example 6 Diethyl{5-acetamido-4-(N²,N³-bis(tert-butoxycarbonyl))guanidino-6-(1,2,3-triacetoxy)propyl-4,5,6-trihydropyran-2-yl}phosphonate(8)

A solution of azide 7 (350 mg, 0.71 mmol) in ethanol (25 mL) washydrogenated with Lindlar catalyst (30 mg) under an atmosphere ofhydrogen. The mixture was stirred for 5 h, filtered through a pad ofCelite, and washed with ethanol. The filtrate was concentrated underreduced pressure to give a colorless foam (278 mg). The crude amineproduct was dissolved in anhydrous CH₂Cl₂ (30 mL) and treated with1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea (247 mg, 0.85 mmol)and Et3N (230 μL, 1.7 mmol). The mixture was cooled to 0° C., and HgCl₂(231 mg, 0.85 mmol) was added slowly. The suspension was warmed to roomtemperature and stirred for 12 h. After which the mixture was dilutedwith EtOAc and filtered through a pad of Celite. The filtrate wasconcentrated and purified by flash column chromatography (EtOAc) toafford guanidine 8 (442 mg, 83% yield) as a colorless foam. TLC (EtOAc)R_(f)=0.45; (α)_(D) ²⁰=+18.5 (c=0.88, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ11.32 (1H, s), 8.48 (1H, d, J=8.5 Hz), 6.12 (1H, d, J=8.5 Hz), 5.71 (1H,dd, J=10.3, 2.0 Hz), 5.35 (1H, d, J=6.6 Hz), 5.23 (1H, td, J=6.5, 2.7Hz), 5.10-5.06 (1H, m), 4.37 (1H, dd, J=12.5, 2.8 Hz), 4.25-4.20 (2H,m), 4.19-4.12 (2H, m), 4.12-4.05 (3H, m), 2.09 (3H, s), 2.06 (3H, s),2.02 (3H, s), 1.85 (3H, s), 1.46 (9H, s), 1.45 (9H, s), 1.36 (3H, t,J=7.1 Hz), 1.31 (3H, t, J=7.1 Hz); ¹³C NMR (150 MHz, CDCl₃) δ 171.0 (C),170.5 (C), 170.1 (C), 169.8 (C), 162.7 (C), 157.2 (C), 152.6 (C), 147.4(C, d, ¹J_(c-p)=224 Hz, C-1), 114.2 (CH, d, ²J_(c-p)=23.3 Hz, C-2), 83.9(C), 79.8 (C), 77.9 (CH, d, ³J_(c-p)=9.3 Hz), 70.1 (CH), 67.4 (CH), 63.4(CH₂, d, ²J_(c-p)=5.7 Hz, POCH₂), 63.0 (CH₂, d, ²J_(c-p)=5.7 Hz, POCH₂),62.1 (CH₂, C-8), 49.0 (CH, d, ³J_(c-p)=15.2 Hz), 48.1 (CH, C-4), 28.2(CH₃, 3×), 28.0 (CH₃, 3×), 23.1 (CH₃), 20.9 (CH₃), 20.8 (CH₃), 20.7(CH₃), 16.29 (CH₃, POCH₂CH₃), 16.25 (CH₃, POCH2CH₃); HRMS calcd forC₃₁H₅₀N_(4O15)P (M⁺−H): 749.3010. found: m/z 749.3172.

Example 7(5-Acetamido-4-amino-6-(1,2,3-hydroxy)propyl-4,5,6-trihydropyran-2-yl)phosphonic acid (1a)

A solution of diethyl phosphonate 7 (80 mg, 0.15 mmol) in anhydrousCH₂Cl₂ (4 mL) at 0° C. was treated with bromotrimethylsilane (0.12 mL,0.87 mmol). After stirring for 24 h at 0° C., MeOH (2 mL) was added, andthe mixture was concentrated under reduced pressure. The residue wasdissolved in anhydrous MeOH (5 mL) and treated with sodium methoxide(5.4 M solution in MeOH, 0.9 mL, 4.86 mmol). After stirring for 1 h atroom temperature, the mixture was filtered through Dowex 50WX8 (H⁺form), and then concentrated under reduced pressure. The residue wasdissolved in MeOH (5 mL) and subjected to hydrogenation (1 atm) in thepresence of Lindlar's catalyst (20 mg) at room temperature. After 3 h,the mixture was filtered through a pad of Celite, and rinsed with MeOH.The filtrate was concentrated, and the residual solids were washed withEt_(2O) (3×10 mL) to afford the phosphonate 1a.

Example 8(5-Acetamido-4-guanidino-6-(1,2,3-hydroxy)propyl-4,5,6-trihydropyran-2-yl)phosphonic acid (1b)

A solution of diethyl phosphonate 8 (130 mg, 0.17 mmol) in anhydrousCH₂Cl₂ (4 mL) was treated with bromotrimethylsilane (0.13 mL, 0.94 mmol)at 0° C., and the reaction mixture was stirred for 24 h at 0° C. MeOH (2mL) was added under vigorous stirring. After 30 min, the solution wasevaporated under reduced pressure and the residue as a solution inanhydrous MeOH (5 mL) was treated with a 5.4 M solution of sodiummethoxide in methanol (1 mL, 5.4 mmol). After stirring for 1 h at roomtemperature, the solution was filtered through Dowex 50WX8 (H⁺ form) andsubjected to lyophilisation. The residual pale yellow solid was washedwith Et_(2O) (3×20 mL) to afford the phosphonate 1b as a white solid.

Example 9(5-Acetamido-4-amino-6-(1,2,3-hydroxy)propyl-4,5,6-trihydropyran-2-yl)phosphonic acid monoethyl ester (1c)

Under an atmosphere of argon, a solution of PMe₃ (1.4 mL, 1.4 M in THF)was added dropwise to a solution of azide 7 (148 mg, 0.28 mmol) inanhydrous THE (5 mL) at 0° C. The mixture was stirred at roomtemperature for 19 h. Et₃N (0.5 mL) and H_(2O) (0.5 mL) was added, andthe mixture was stirred for another 30 min. The mixture was concentratedunder reduced pressure, and purified by silica gel column chromatography(MeOH/CH₂Cl₂, 6:94 to 10:90) to afford an amine product (112 mg, 79%yield).

The amine product (110 mg, 0.22 mmol) was dissolved in EtOH (2 mL), anda solution of NaOEt (490 μL, 2.68 M in EtOH) was added dropwise. Themixture was stirred for 2 days and monitored by TLC. After neutralizedby Dowex SOW resin, the filtrate was concentrated under reducedpressure, and subjected to silica gel column chromatography(n-PrOH/H_(2O), 7:3). The appropriate fractions were collected andconcentrated under reduced pressure. The residue was treated with 1 MHCl (2 mL), and then concentrated in vacuo to give phosphonate monoester1c (50 mg, 60% yield). C₁₂H₂₃N_(2O8)P: ¹H NMR (600 MHz, D_(2O)) δ 5.48(1H, dd, J=9.4, 7.6 Hz), 4.38-4.33 (2H, m), 4.18-4.17 (1H, m), 3.98-3.93(2H, m), 3.91-3.86 (2H, m), 3.69-3.64 (2H, m), 2.07 (3H, s), 1.26 (3H,t, J=7.1 Hz); ¹³C NMR (150 MHz, D_(2O)) δ 174.7, 153.3 (d,¹J_(C-P)=208.8 Hz), 104.3 (d, ²J_(C-P)=22.5 Hz), 75.4 (d, ³J_(C-P)=8.0Hz), 69.6, 67.6, 62.9, 62.2 (d, ²J_(C-P)=5.1 Hz), 49.9 (d, ³J_(C-P)=13.4Hz), 45.8, 22.1, 15.8 (d, ³J_(C-P)=5.7 Hz); HRMS calcd forC₁₂H₂₂N_(2O8)P: 353.1108. found: m/z 353.1198 [M−H]⁻.

Example 10(5-Acetamido-4-guanidino-6-(1,2,3-hydroxy)propyl-4,5,6-trihydropyran-2-yl)phosphonic acid monoethyl ester (1d)

Compound 8 (52 mg, 0.07 mmol) was dissolved in EtOH (1 mL) and asolution of NaOEt (209 μL, 2.68 M in EtOH) was added dropwise. Themixture was stirred for 4 days and monitored by TLC. After neutralizedby Dowex 50W resin, the filtrate was concentrated under reducedpressure, and subjected to silica gel column chromatography(n-PrOH/H_(2O), 8:2). The appropriate fractions were collected andconcentrated under reduced pressure. The residue was treated with 1 MHCl (2 mL), and then concentrated in vacuo to give phosphonate monoester1d (9 mg, 30% yield). C₁₃H₂₅N_(4O8)P: ¹H NMR (600 MHz, D_(2O)) δ 5.41(1H, dd, J=9.4, 7.5 Hz), 4.42-4.40 (1H, m), 4.35 (1H, d, J=10.6 Hz),4.21 (1H, t, J=9.9 Hz), 3.97-3.93 (2H, m), 3.90-3.85 (2H, m), 3.67-3.62(2H, m), 2.01 (3H, s), 1.25 (3H, t, J=7.1 Hz); ¹³C NMR (150 MHz, D_(2O))δ 174.3, 156.9, 151.3 (d, ¹J_(C-P)=211.2 Hz), 108.2 (d, ²J_(C-P)=21.6Hz), 75.8, (d, ³J_(C-P)=8.6 Hz), 69.7, 67.9, 62.9, 62.1 (d, ²J_(C-P)=4.8Hz), 50.9 (d, ³J_(C-P)=13.7 Hz), 47.8, 21.9, 15.8 (d, ³J_(C-P)=5.7 Hz).

Example 11 Synthesis of Zanamivir Biotin Conjugate

FIG. 3 shows the synthesis of zanamivir-biotin conjugate 9. Theazide-annexed biotin derivative 11 was prepared by condensation ofbiotin (10) with 5-azido-1-pentylamine in the presence of triethylamineand benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP). (J. W. Lee, S. I. Jun, K. Kim, Tetrahedron Lett. 42, 2709(2001)). On the other hand, zanamivirp-nitrophenyl carbonate 12,prepared from sialic acid according to the known procedure, (M. Chandleret al., J. Chem. Soc., Perkin Trans. 1: 1173 (1995); L. Ying, J.Gervay-Hague, ChemBioChem 6, 1857 (2005)) was coupled withpropargylamine to afford the zanamivir derivative 13 with an alkynylhinge. (W.-H. Wen et al., J. Med. Chem. 52, 4903 (2009)). The subsequent1,3-dipolar addition (click reaction; V. V. Rostovtsev et al., Angew.Chem., Int. Ed. 41, 2596 (2002); B.-Y. Lee et al., Tetrahedron Lett. 47,5105 (2006)) between the azide-annexed biotin derivative 11 and thealkynyl zanamivir derivative 13 was conducted in a mixed solvent ofCH₂Cl₂/H_(2O) (1:1) to give the desired zanamivir-biotin conjugate 9 in88% overall yield after removal of the protecting groups.

Example 12 Biotin Derivative 11

A solution of PPh₃ (5.0 g, 19.1 mmol) and 1,5-diazidopentane (3.36 g,21.8 mmol), prepared from the substitution reaction of1,5-dibromopentane with NaN3, was vigorously stirred with 5% aqueous HCl(22 mL) in EtOAc/Et2O (v/v=1:1, 35 mL) at room temperature for 24 h togive 5-azido-1-pentylamines1 (1.60 g, 59%). (J. W. Lee, S. I. Jun, K.Kim, Tetrahedron Lett. 42, 2709 (2001).)

A sample of biotin (10, 136 mg, 0.56 mmol) was stirred with5-azido-1-pentylamine (11.86 mg, 0.69 mmol),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP, 360 mg, 0.69 mmol) and Et3N (0.116 mL, 0.84 mol) in DMF solution(5 mL) at room temperature for 3 h. The mixture was concentrated underreduced pressure, and washed with H2O. The residue was purification bysilica gel chromatography (CH2Cl2/MeOH=15:1) to give the azide-annexedbiotin derivative 11 (180 mg, 92%). C15H26N6O2S; colorless solid, mp121-122° C.; [α]19D+87.6 (c 0.0275, MeOH); IR vmax (neat) 3297, 2924,2100, 1698, 1647 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.34 (1H, t, J=5.2 Hz),6.40 (1H, s), 6.34 (1H, s), 4.30-4.27, (1H, m), 4.12-4.09 (1H, m), 3.29(2H, t, J=6.8 Hz), 3.10-3.05 (1H, m), 3.03-2.98 (2H, m), 2.80 (1H, dd,J=12.4, 5.2 Hz), 2.56 (1H, d, J=12.4 Hz), 2.03 (2H, t, J=7.2 Hz),1.64-1.22 (12H, m); ¹³C NMR (100 MHz, DMSO) δ 171.5, 162.4, 61.0, 59.1,55.4, 50.6, 39.8, 38.1, 35.2, 28.7, 28.3, 28.1, 28.0, 25.4, 23.6;ESI-HRMS calculated for C₁₅H₂₇N_(6O2)S: 355.1916. found: m/z 355.1913[M+H]⁺.

Example 13 Zanamivir-Biotin Conjugate 9

A solution of the azide-annexed biotin derivative 11 (100 mg, 0.28 mol),and the alkynyl-hinged zanamivir derivative 13 (188 mg, 0.28 mmol) wasstirred with CuSO₄.5H_(2O) (10 mg, 0.04 mmol) and sodium ascorbate (25mg, 0.13 mmol) in CH₂Cl₂/H_(2O) (6 mL, v/v=1:1) at room temperature for8 h. The aqueous layer was extracted with CH₂Cl₂. The organic layer wascombined, dried over MgSO₄, filtered, and concentrated by rotaryevaporation under reduced pressure. The residue was purified by columnchromatography (CH₂Cl₂/MeOH=20:1 to 10:1) to afford a zanamivir-biotinconjugate containing protective groups (270 mg, 95%). C₄₅H71N_(11O14)S;TLC (CH₂Cl₂/MeOH=9:1) R_(f)=0.29; colorless solid, mp 157-158° C.; [α]²²_(D)+16.6 (c 0.5, CH₂Cl₂); IR vmax (neat) 2930, 1727, 1689, 1643, 1612cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 11.37 (1H, s), 8.20 (1H, d, J=7.6 Hz),8.04 (1H, d, J=9.2 Hz), 7.95 (1H, s), 7.76-7.70 (2H, m), 6.40 (1H, s),6.33 (1H, s), 5.81 (1H, d, J=2.0 Hz), 5.17 (1H, d, J=5.6 Hz), 4.79 (1H,t, J=7.6 Hz), 4.36 (1H, d, J=11.6 Hz), 4.30-4.21 (4H, m), 4.15-4.10 (3H,m), 4.04-3.97 (2H, m), 3.86 (1H, dd, J=8.8, 5.6 Hz), 3.71 (3H, s),3.10-3.05 (1H, m), 3.00-2.95 (2H, m), 2.80 (1H, dd, J=12.4, 5.2 Hz),2.56 (1H, d, J=12.4 Hz), 2.02 (2H, t, J=7.2 Hz), 1.80-1.77 (2H, m), 1.74(3H, s), 1.64-1.19 (10H, m), 1.45 (9H, s), 1.39 (9H, s), 1.25 (6H, s);¹³C NMR (100 MHz, CDCl₃) δ 173.0, 170.7, 163.7, 162.8, 161.8, 156.7,155.6, 152.4, 145.4, 144.6, 122.9, 110.1, 108.6, 83.6, 79.6, 77.5, 77.2,75.1, 69.9, 65.7, 61.9, 60.2, 55.8, 53.5, 52.5, 50.0, 49.5, 47.4, 40.6,38.9, 37.0, 35.8, 29.6, 28.6, 28.3 (3×), 28.1 (3×), 26.5, 25.6, 25.5,23.5, 23.2; ESI-HRMS calcd for C₄₅H₇₂N_(11O14)S: 1022.4981. found: m/z1022.4986 [M+H]⁺.

A sample of the protected zanamivir-biotin conjugate (34 mg, 0.033 mmol)was treated with aqueous NaOH (1 M, 1 mL) in MeOH (1 mL) at roomtemperature for 15 min. The mixture was neutralized with Dowex 50Wx8(H⁺), filtered, and concentrated under reduced pressure. The residue wasthen stirred with trifluoroacetic acid (TFA, 1 mL) in CH₂Cl₂ (1 mL) atroom temperature for 1.5 h. The mixture was evaporated under reducedpressure, and H_(2O) (1 mL) was added at room temperature. Afterstirring for 10 min, the mixture was concentrated under reducedpressure, and purified by chromatography on a Sephadex G-10 column(eluent: 0.1% TFA in H_(2O)) to give the desired zanamivir-biotinderivative 9 (24 mg, 93%). C₃₁H₄₉N_(11O10)S; colorless solid, mp170-172° C.; IR v_(max) (neat) 3355, 2937, 1675 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 7.92 (1H, s), 5.96 (1H, s), 4.98 (1H, d, J=8.4 Hz), 4.61-4.57(2H, m), 4.47-4.39 (5H, m), 4.33-4.29 (1H, m), 4.16 (1H, t, J=9.2 Hz),4.06-4.04 (1H, m), 3.65 (1H, d, J=9.6 Hz), 3.47 (1H, dd, J=6.4, 12.0Hz), 3.31 (1H, m), 3.20-3.10 (2H, m), 2.98 (1H, dd, J=4.8, 13.2 Hz),2.77 (1H, d, J=13.2 Hz), 2.22 (2H, t, J=7.2 Hz), 1.97 (3H, s), 1.93-1.90(2H, m), 1.73-1.51 (6H, m), 1.38-1.22 (4H, m); ¹³C NMR (100 MHz, CDCl₃)δ 176.4, 173.8, 165.3, 164.9, 162.9 (CO₂ of TFA, q, J=35.0 Hz), 157.0,156.5, 124.1, 116.5 (CF₃ of TFA, q, J=288.1 Hz), 109.1, 75.9, 70.2,69.0, 62.6, 62.4, 60.5, 55.7, 51.3, 50.8, 47.4, 40.0, 39.2, 36.0, 35.8,29.4, 29.3, 28.2, 28.1, 28.0, 25.5, 23.3, 22.2; ESI-HRMS calculated forC₃₁H₅₀N_(11O10)S: 768.3463. found: m/z 768.3458 [M+H]⁺.

Example 14 Compound 14

To a solution of carbonate 12 (0.25 g, 0.33 mmol),4-dimethylaminopyridine (DMAP, 60 mg, 0.50 mmol) in pyridine (2 mL) wasadded an amine H₂N(CH₂CH₂O)₃CH₂CH₂N₃ (0.14 g, 0.66 mmol). The mixturewas stirred for 40 h at room temperature under an atmosphere of N₂, andthen extracted with HCl (10 mL of 1 M aqueous solution) and EtOAc (50mL). The organic layer was washed with brine (30 mL) and saturatedNaHCO₃(a_(q)) (30 mL), dried over MgSO4, and concentrated under reducedpressure. The residue was purified by column chromatography (silica gel,EtOAc/hexane=1:1) to give carbamate 14 (0.2 g, 74%). C₃₅H₅₈N_(8O15); 1HNMR (CDCl₃, 600 MHz) δ 11.41 (1H, s), 8.45 (1H, d, J=9 Hz), 6.08 (1H, d,J=9 Hz), 5.91 (1H, s), 5.45 (1H, t, J=6 Hz), 5.26 (1H, t, J=10 Hz), 5.22(1H, d, J=6 Hz), 4.41 (1H, d, J=9 Hz), 4.38 (1H, q, J=10, 6 Hz),4.10-4.14 (2H, m), 3.81 (3H, s), 3.60-3.77 (12H, m), 3.56-3.57 (1H, m),3.41 (2H, t, J=5 Hz), 3.37 (2H, d, J=5 Hz), 1.92 (3H, s), 1.50 (18H, s),1.41 (3H, s), 1.37 (3H, s); 13C NMR (CDCl₃) δ 170.6, 163.0, 161.9,156.9, 155.6, 152.7, 145.2, 132.1, 132.0, 131.9, 128.5, 128.4, 109.8,108.9, 83.6, 79.6, 77.4, 74.5, 70.6, 70.5, 70.2, 70.0, 69.7, 66.0, 60.4,52.4, 50.6, 48.7, 48.3, 41.0, 28.2, 28.0, 26.6, 25.4, 23.1; ESI-HRMScalcd for C35H59N8O15: 831.4100. found: m/z 831.4134 [M+H]⁺.

Example 15 Zanamivir-FITC Conjugate 15

To a solution of azide 14 (80 mg, 0.096 mmol) in EtOH (1 mL) was addedPd(OH)2 (9 mg, 0.058 mmol). The mixture was stirred for 1.5 h at roomtemperature under an atmosphere of H₂, and then filtered through a padof Celite by elution with MeOH. The mixture was concentrated underreduced pressure to give an amine product (66 mg). C₃₅H₆₀N_(6O15); ¹HNMR (CDCl₃, 600 MHz) δ 11.36 (1H, s), 8.40 (1H, d, J=8 Hz), 6.18 (1H, d,J=9 Hz), 6.04 (1H, s), 5.87 (1H, s), 5.17-5.22 (2H, m), 4.32-4.39 (2H,m), 3.98-4.10 (3H, m), 3.75 (3H, s), 3.55-3.72 (12H, m), 3.31 (4H, s),2.89 (1H, s), 2.73 (1H, d, J=7 Hz), 1.88 (3H, s), 1.45 (18H, s), 1.36(3H, s), 1.32 (3H, s).

Under an atmosphere of N₂, fluorescene thiocyanate (FITC, 37 mg, 0.095mmol) was added to a solution of the above-prepared amine compound inanhydrous THF (1 mL). The mixture was stirred for 4 h at roomtemperature, concentrated under reduced pressure, and purified by columnchromatography (silica gel, CH₂Cl₂/MeOH=92:8) to give a conjugationproduct (50 mg, 52%). C₅₆H₇₁N₇O₂₀S; ¹H NMR (MeOD, 600 MHz) δ 7.80 (1H,s), 7.56-7.59 (2H, m), 7.48 (1H, t, J=8 Hz), 7.17 (1H, d, J=8 Hz), 7.09(1H, d, J=8 Hz), 6.63 (2H, s), 6.56 (2H, d, J=9 Hz), 6.48-6.51 (3H, m),5.86 (1H, d, J=8 Hz), 4.30 (1H, t, J=10 Hz), 4.12-4.23 (2H, m),4.07-4.10 (2H, m), 3.96-4.06 (2H, m), 3.93 (1H, t, J=8 Hz), 3.60-3.77(15H, m), 3.44-3.58 (4H, m), 3.16-3.24 (2H, m), 1.82 (3H, s), 1.40-1.42(18H, m), 1.32 (3H, s), 1.30 (3H, s).

A solution of the above-prepared FITC conjugate (47 mg, 0.039 mmol) inMeOH (1.5 mL) was treated with NaOH (1 mL of 1 M aqueous solution). Themixture was stirred for 1 h at room temperature, neutralized with Dowex50wX8 (H⁺), and filtered. The filtrate was concentrated, and the residuewas treated with trifluoroacetic acid (TFA, 1 mL) in CH₂Cl₂ (1 mL). Themixture was stirred for 1 h, concentrated under reduced pressure, andpurified by chromatography on a Sephadex G-10 column (eluent: 0.1% TFAin H₂O) to give the desired zanamivir-FITC conjugate 15 (10 mg, 56%).C₃₈H₄₁N₇O₁₄S; ¹H NMR (MeOD, 600 MHz) δ 8.31 (1H, s), 7.88 (1H, d, J=8Hz), 7.48-7.57 (3H, m), 7.30 (1H, s), 7.36 (1H, s), 7.13 (2H, d, J=9Hz), 6.00 (1H, s), 4.30-4.32 (1H, m), 4.10-4.12 (2H, m), 3.81-3.97 (2H,m), 3.58-3.80 (14H, m), 3.13-3.28 (3H, m), 1.80 (3H, s).

Example 16 Zanamivir-FITC Conjugate 17

To a solution of alkyne 13 (87 mg, 0.13 mmol) and 10-azidodecanamine (30mg, 0.16 mmol) in t-BuOH (1 mL) and H₂O (1 mL) were added CuSO₄.5H₂O (4mg, 0.016 mmol), sodium ascorbate (18 mg, 0.091 mmol) andtris[(1-benzyl-1H-1,2,3-triazol-1-yl)methyl]amine (TBTA, 8 mg, 0.04mmol). The mixture was stirred for 12 h at room temperature, and thenextracted with CH₂Cl₂ (30 mL) and H₂O (30 mL). The organic layer wasdried over MgSO₄, concentrated under reduced pressure, and purified bycolumn chromatography (silica gel, CH₂Cl2/MeOH=9:1) to give compound 16(53 mg, 50%). C₄₀H₆₇N₁₉O₁₂; yellow solid, mp=116° C. ¹H NMR (CDCl₃, 600MHz) δ 11.38 (1H, s), 8.43 (1H, d, J=8 Hz), 7.84 (1H, br s), 5.88 (1H,d, J=14 Hz), 5.11-5.28 (2H, m), 4.33-4.45 (6H, m), 4.18 (1H, s), 4.08(1H, s), 3.99 (1H, s), 3.76 (3H, s), 1.89 (3H, s), 1.31 (18H, s), 1.23(22H, s).

To a solution of the above-prepared amine (0.1 g, 0.115 mmol) in THF (1mL) and MeOH (2 mL) were added FITC (45 mg, 0.115 mmol) anddiisopropylethylamine (0.038 mL). The mixture was stirred at roomtemperature for 20 h, and then concentrated under reduced pressure togive a conjugation product as red solids. A solution of theabove-prepared conjugation compound in MeOH (1 mL) was stirred with NaOH(1 mL of 1 M aqueous solution) for 1 h at room temperature, neutralizedby Dowex 50wX8 (H⁺), and filtered. After concentration, the residue wasdissolved in CH₂Cl₂ (1 mL) and TFA (1 mL). The mixture was stirred for30 min at room temperature, concentrated under reduced pressure, andpurified by chromatography on a Sephadex G-10 column (eluent: 0.1% TFAin H₂O) to give the desired zanamivir-FITC conjugate 17 (50 mg, 43%).C₄₇H₅₆N₁₀O₁₃S; yellow solid, mp=112° C.; ¹H NMR (d-MeOD, 600 MHz) δ 8.13(1H, d, J=7 Hz), 7.63-7.67 (4H, m), 7.55-7.58 (3H, m), 6.89 (1H, d, J=7Hz), 4.54 (1H, br s), 4.31-4.38 (2H, m), 4.17 (1H, s), 4.00 (1H, br s),3.58-3.68 (12H, m), 3.56 (2H, t, J=5 Hz), 3.42-3.47 (2H, m), 2.00 (3H,s), 1.70-1.72 (4H, m), 1.18-1.36 (12H, m); ESI-HRMS calcd forC₄₇H₅₇N₁₀O₁₃S: 1001.3827. found: m/z 1001.3768 [M+H]⁺.

Example 17 Synthesis of Tamiphosphor-Biotin Conjugate

FIG. 4 shows the synthesis of tamiphosphor-biotin conjugate 24. Theiodide compound 18 was prepared according to the previously reportedprocedure. [Shie, J.-J., et al. Angew. Chem. Int. Ed. 2008, 47, 5788.]Di[4-(trimethylsilyl)but-3-yn-1-yl] Dialkyl phosphite 17 was prepared bythe substitution reaction of PCl₃ with two equivalents of4-(trimethylsilyl)but-3-yn-1-ol. Phosphonylation of 18 with phosphite 19was achieved by the catalysis of Pd(PPh₃)₄ to afford the phosphonate 20in 64% yield. After removal of the Boc group, the amine intermediate wastreated with N,N′-di-Boc-N″-trifluoromethanesulfonyl-guanidine, followedby removal of trimethylsilyl group with KF, to give compound 21. Theclick reaction of 21 with 5-azidopentanamine, followed by treatment withKOH, afforded phosphonate monoester 22. The coupling reaction of 20 withbiotin-OSu (23) culminated in the desired tamiphosphor-biotin conjugate24 after removal of the protecting Boc groups.

Example 18 Compound 20

A mixture of iodine 18 (100 mg, 0.21 mmol), phosphite 19 (85 mg, 0.26mmol) and diisopropylethylamine (100 mg, 0.64 mmol) in anhydrous toluene(2.1 mL) was deoxygenated by bubbling with nitrogen for 10 min, and thenadded to tetrakis(triphenylphosphine)palladium(0) (10 mg, 8.6 μmol) thatwas placed in a round bottomed flask under nitrogen atmosphere. Theresulting solution was gradually heated to 90° C. and maintained at thistemperature for 12 h. The reaction mixture was filtered through a pad ofCelite, and the filtrate was evaporated under reduced pressure to giveyellow foam (110 mg), which was purified by flash chromatography on asilica gel column [EtOAc/hexane=1:1 to EtOAc] to afford phosphonate 20(92 mg, 64%). C₃₂H₅₇N₄O₉P; yellow oil; TLC (EtOAc/hexane, 1:1)R_(f)=0.29; 1H NMR (400 MHz, CDCl₃) δ 6.59 (1H, d, J=22.0 Hz), 5.85 (1H,d, J=9.2 Hz), 5.04 (1H, d, J=9.2 Hz), 3.97-4.11 (5H, m), 3.89 (1H, br),3.75-3.81 (1H, m), 3.29-3.32 (1H, m), 2.57-2.61 (1H, m), 2.59 (4H, t,J=1.2 Hz), 2.20 (1H, td, J=10.0, 3.2 Hz), 2.00 (3H, s), 1.43-1.51 (4H,m), 1.39 (9H, s), 0.83-0.88 (6H, m), 0.07-0.21 (18H, m); ¹³C NMR (100MHz, CDCl₃) δ 170.2, 155.6, 142.1, 127.2, 101.2, 87.0, 82.2 (2×), 79.7,76.0, 63.7 (2×), 54.8, 49.3, 49.1, 31.4, 28.6 (3×), 26.4, 25.9, 23.7,22.6, 22.5, 10.1, 9.9, 0.5 (6×); ³¹P NMR (162 MHz, CDCl₃) δ 18.0.

Example 19 Compound 21

A solution of phosphonate 20 (92 mg, 0.14 mmol) in anhydrous CH₂Cl₂ (1.0mL) was cooled to 0° C. in an ice bath, and trifluoroacetic acid (0.16mL, 2.1 mmol) was added. The mixture was stirred for 1 h at roomtemperature, and concentrated under reduced pressure, and then dissolvedin anhydrous CH₂Cl₂ (1.0 mL).N—N′-Di-Boc-N″-trifluoromethanesulfonylguanidine (67 mg, 0.21 mmol) andtriethylamine (0.06 mL, 0.41 mmol) were added. The mixture was stirredat room temperature for 3 h, and extracted with 1 M HCl (5 mL) andCH₂Cl₂ (5 mL×3). The organic layer was dried over MgSO₄, filtered,concentrated, and purified by flash chromatography on a silica gelcolumn (EtOAc/hexane=1:1) to yield a guanidine derivative.

To a solution of the guanidine compound in MeOH/H₂O (1.2 mL/1.2 mL, v/v)was added KF (71 mg, 1.2 mmol). The mixture was stirred at roomtemperature for 24 h, and then concentrated under reduced pressure. Themixture was extracted with CH₂Cl₂ (5 mL×3) and H₂O (5 mL). The organiclayer was dried over MgSO₄, filtered, concentrated, and purified byflash chromatography on a silica gel column (EtOAc/hexane=1:1) to yieldcompound 21 (63 mg, 69%). C₃₂H₅₁N₄O₉P; colorless oil; TLC (EtOAc/hexane,1:1) R_(f)=0.24; [α]_(D) ²²=−27.47 (c=1, CH₂Cl₂); IR (film) v_(max)3450, 2923, 2018, 1925, 1870, 1720, 1626, 1342, 1249, 1203; ¹H NMR (400MHz, CDCl₃) δ 11.34 (1H, s), 8.56 (1H, d, J=8.0 Hz), 6.66 (1H, d, J=22.4Hz), 6.28 (1H, d, J=9.2 Hz), 4.34-4.40 (1H, m), 4.06-4.16 (5H, m),3.96-4.04 (1H, m), 3.29-3.33 (1H, m), 2.65-2.71 (1H, m), 2.59-2.61 (4H,m), 2.26-2.40 (1H, m), 2.03 (1H, s), 2.00 (1H, s), 1.90 (3H, s),1.00-1.78 (22H, m), 0.83-0.90 (6H, m); ³¹P NMR (162 MHz, CDCl₃) δ 18.0;HRMS calcd for C₃₂H₅₂N₄O₉P: 667.3472. found: m/z 667.3450 [M+H]⁺.

Example 20 Tamiphosphor-Biotin Conjugate 24

To a solution of 5-azido-1-pentanamine azide (46 mg, 0.36 mmol) andcompound 19 (120 mg, 0.18 mmol) in t-BuOH/H₂O (0.6 mL, v/v=1:1) wasadded tetrakis(acetonitrile)copper(I) phosphorus hexafluoride. Themixture was stirred at room temperature for 12 h, concentrated byreduced pressure, and purified on a by RP-18 reversed-phase column withelution of MeOH/H₂O (1:9 to 9:1). The crude product of triazole compoundwas dissolved in 1,4-dioxane (1.0 mL), and added 1 M KOH_((aq)) (1.0mL). The solution was stirred at 25° C. for 120 h (monitored by ¹H NMR),and added Dowex 50Wx8 to neutralize the solution. The mixture wasfiltered and concentrated under reduced pressure. The crude product (10mg, 0.013 mmol) was dissolved in anhydrous DMF (0.1 mL), and biotin-OSu(4.5 mg, 0.013 mmol) and diisopropylethylamine (4.2 mg, 0.026 mmol) wereadded. The mixture was stirred at room temperature for 3 h, andconcentrated under reduced pressure. The residue was dissolved in MeOH(0.5 mL), cooled to 0° C. in an ice bath, and added trifluoroacetic acid(0.16 mL, 2.1 mmol). The mixture was stirred for 1 h at roomtemperature, concentrated under reduced pressure, and purified on aRP-18 reversed-phase column with elution of MeOH/H₂O (1:9 to 9:1) togive the title compound (40 mg, 30% overall yield). C33H₅₇N₁₀O₇PS;yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (1H, s), 6.39 (1H, d,J=19.6 Hz), 4.90 (1H, t, J=5.2 Hz), 4.38 (1H, t, J=7.2 Hz), 4.29-4.32(1H, m), 4.00 (2H, m), 3.77-3.90 (2H, m), 3.40-3.48 (2H, m), 3.15-3.26(2H, m), 3.00 (1H, t, J=5.2 Hz), 2.91-2.95 (2H, m), 2.71 (1H, d, J=12.8Hz), 2.53-2.62 (2H, m), 2.23 (2H, t, J=7.2 Hz), 1.90-2.03 (5H, m),1.29-1.79 (16H, m), 0.86-0.97 (6H, m); ³¹P NMR (162 MHz, CDCl₃) δ 13.6;HRMS negative mode calcd for C₃₃H₅₆N₁₀₀7PS: 768.3723. found: m/z768.3723 [M−H]⁻.

Example 21 Compound 25

To a solution of di(5-azidopentyl)amine (0.12 g, 0.49 mmol) in anhydrousDMF (5 mL) of were added biotin (0.12 g, 0.41 mmol), PyBOP (0.25 g, 0.49mmol) and Et₃N (0.23 mL, 1.64 mmol). The mixture was stirred for 22 h atroom temperature under argon, and then concentrated under reducedpressure. The residue was purified by column chromatography (silica gel,CH₂Cl₂/MeOH=13:1) to give biotin-diazide compound 25 (0.15 g, 79%).C₂₀H₃₅N₉O₂S; pale yellow oil; ¹H NMR (CDCl₃, 600 MHz) δ 4.48 (dd, J=7, 5Hz, 1H), 4.29 (dd, J=7, 5 Hz, 1H), 3.28 (t, J=7 Hz, 4H), 3.25 (t, J=7Hz, 4H), 3.20 (t, J=7 Hz, 2H), 2.89 (dd, J=12, 5 Hz, 1H), 2.71 (d, J=12Hz, 1H), 2.29 (t, J=7 Hz, 2H), 1.84 (br s, 3H), 1.50-1.67 (m, 8H),1.43-1.44 (m, 2H), 1.32-1.38 (m, 4H); ¹³C NMR (CDCl₃, 150 MHz) δ 172.4,163.2, 61.8, 60.1, 55.3, 51.2, 47.0, 46.2, 45.6, 40.5, 32.5, 28.7, 28.6,28.3, 27.3, 26.4, 25.1, 24.1. ESI-HRMS calcd for C₂₀H₃₆N₉O₂S: 466.2707.found: m/z 466.2703 [M+H]⁺.

Example 22 Dizanamivir-Biotin Conjugate 26

To a solution of biotin-diazide 25 (36 mg, 0.075 mmol) in t-BuOH (1 mL)and H₂O (1 mL) were added CuSO₄.5H₂O (4 mg, 0.015 mmol), sodiumascorbate (10 mg, 0.045 mmol), and TBTA (8 mg, 0.015 mmol). The mixturewas stirred for 5 min, and the alkynyl-hinged zanamivir derivative 13(0.1 g, 0.15 mmol) was added. The mixture was stirred for 12 h at roomtemperature, extracted with CH₂Cl₂ (20 mL) and H₂O (20 mL), dried overMgSO₄, and concentrated under reduced pressure. The residue was purifiedby column chromatography (silica gel, CH₂Cl₂/MeOH=9.5:1) to give thecoupling product (64 mg, 47%). ¹H NMR (CDCl₃, 600 MHz) δ 1.22 (s, 12H),1.30 (s, 36H), 1.62 (s, 4H), 1.79 (s, 2H), 1.89 (s, 3H), 1.94 (s, 3H),2.23 (t, J=7 Hz, 2H), 2.74 (d, J=12 Hz, 1H), 2.88 (d, J=12 Hz, 1H),3.10-3.16 (m, 4H), 3.23 (s, 2H), 3.75 (s, 6H), 3.98 (t, J=7 Hz, 2H),4.06 (t, J=8 Hz, 2H), 4.15 (t, J=10 Hz, 2H), 4.28-4.43 (m, 14H), 5.16(s, 2H), 5.23 (t, J=6 Hz, 2H), 5.35 (s, 1H), 5.70 (s, 1H), 5.86 (d, J=8Hz, 2H), 6.04 (s, 1H), 6.28 (s, 1H), 6.54 (d, J=9 Hz, 1H), 6.63 (d, J=9Hz, 1H), 7.79 (s, 2H), 8.42 (d, J=8 Hz, 2H), 11.38 (s, 2H).

The above-prepared coupling product (60 mg, 0.033 mmol) in MeOH (1 mL)was stirred with NaOH (1 mL of 1 M aqueous solution) for 1 h at roomtemperature, neutralized with Dowex 50Wx8 (H⁺), and filtered. Afterconcentration, the residue was dissolved in CH₂Cl₂ (1 mL) and TFA (1mL). The mixture was stirred for 1 h at room temperature, concentratedunder reduced pressure, and purified by chromatography on a SephadexG-10 column (eluent: 0.1% TFA in H₂O) to give the dizanamivir-biotinconjugate 26 (50 mg, 43%). C₅₂H₈₁N₁₉O₁₈S; ¹H NMR (CDCl₃, 600 MHz) δ1.29-1.33 (m, 6H), 1.45 (br s, 2H), 1.56-1.61 (m, 8H), 1.75 (br s, 1H),1.84-1.87 (m, 1H), 1.97-2.04 (m, 12H), 2.36 (br s, 2H), 2.73 (d, J=12Hz, 1H), 2.94 (d, J=12 Hz, 1H), 3.51 (br s, 2H), 3.65 (d, J=10 Hz, 2H),3.69 (s, 2H), 3.78 (s, 4H), 4.00 (br s, 2H), 4.19 (br s, 2H), 4.37-4.45(m, 11H), 4.55 (br s, 4H), 4.68 (d, J=8 Hz, 4H), 5.91 (s, 2H), 7.57 (s,1H), 8.05 (s, 2H); ESI-HRMS calcd for C₅₂H₈₂N₁₉O₁₈S: 1292.5800. found:m/z 1292.5916 [M+H]⁻.

Example 23 Cells, Viruses, and Biological Reagents

Both the MDCK and the 293T cells and two influenza viruses: A/Aichi/2/68(H3N2) and B/Lee/40 were obtained from ATCC (Manassas, Va., USA),respectively. Influenza viruses A/WSN/1933 (H1N1), A/Udorn/307/1972(H3N2), A/PR/8/1934 (H1N1), A/Taiwan/3446/2002 (H3N2), influenzaB/Taiwan/7064/2004 isolates were from Dr. Shin-Ru Shih's lab (Chang GungUniversity, Taiwan), and ANietnam/1194/2004 RG14 (H5N1),A/California/7/2009 (H1N1), A/Brisbane/10/2007 (H1N1) andA/Brisbane/10/2007 (H3N2) were from Dr. Jia-Tsrong Jan's lab (GenomicsResearch Center, Academia Sinica, Taiwan). The oseltamivir resistant WSNmutant was selected by 6 passages in MDCK cells with gradually increasedOC (oseltamivir carboxylate) concentrations. This mutant influenza growswell in the presence of 1 μM OC and carries a single H274Y mutation atits NA gene confirmed by sequence analysis. Other Taiwan clinical H1N1isolates were obtained from the influenza collection center of Centerfor Disease Control (Taipei, Taiwan) and are described in Table 1. SevenS-OIV isolates were also obtained from Center for Disease Control(Taipei, Taiwan) and were coded as #1 (A/Taiwan/T1941/2009), #2(A/Taiwan/T1338/2009), #3 (A/Taiwan/T1339/2009), #4(A/Taiwan/6662/2009), #5 (A/Taiwan/6663/2009), #6 (A/Taiwan/7717/2009),and #7 (A/Taiwan/7855/2009). The antibodies for influenza NP and thefluorescein-labeled secondary antibody were purchased from Chemicon Inc.(Billerica, Mass., USA) and Sigma (St. Louis, Mo., USA), respectively.Anti-WSN rabbit antibody was prepared in the lab using inactivated WSNinfluenza as the antigen. The anti-HA antibody (Abcam, Cambridge, Mass.,USA) was purchased from Interlab Ltd. (Taipei, Taiwan).

Example 24 Development of the OC Susceptibility Assay UsingNeuraminidase Expressing Cells

The neuraminidase inhibitory activities of zanamivir and ZB werecompared and it was found that ZB is still a very potent inhibitor forneuraminidase with an IC₅₀ of 7 nM although it is slightly less activethen zanamivir with an IC₅₀ of 2 nM. Functional neuraminidase expressedon the cell surface could be useful as a model system for ZB binding andOC competition for OC susceptibility assessments. The cDNA for theneuraminidase of A/Hanoi/30408/2005 (H5N1) was cloned in an expressionplasmid and used to transfect 293T cells for the expression of the wildtype (274H) neuraminidase. The expressed neuraminidase was found to belocated mainly on the cell surface allowing the binding and labelingusing ZB (FIG. 10). The neuraminidase cDNA was mutagenized for theexpression of the OC resistant 274Y mutant enzyme. Both the wild typeand the mutagenized cDNA were used to generate stable 293T cell linesfor the expression of the 274H and 274Y neuraminidases, respectively.Mixtures of these two cells at different ratios were incubated with ZBor ZB plus excess OC and then decorated with APC conjugated streptavidinto determine the population of cells that bind ZB by flow cytometryanalyses. FIG. 11 shows that ZB bound 274H and 274Y expressing cells tosimilar extents. In the presence of excess OC, ZB binding was completelyblocked in cells expressing 274H neuraminidase, whereas ZB binding tothe 274Y expressing cells was virtually unaltered (FIG. 12). Theseresults prove the principle that ZB binding can be used to differentiateOC-sensitive and OC-resistant neuraminidases by OC competition.

Example 25 Determination of OC Susceptibility Using Influenza VirusInfected Cells

The results of the competitive OC inhibition on ZB binding using NAexpressing cells had prompted us to define the concentrations of ZB andOC to assess the OC susceptibility of influenza viruses. MDCK cellsinfected with either OC susceptible or OC resistant WSN viruses wereused to test feasibility of the OC susceptibility assessment usingvaried ZB and OC concentrations. FIG. 13A shows OC competition of ZBbinding to influenza virus infected MDCK cells. By labeling influenzainfected cells with either 10 or 50 nM ZB, OC susceptibility ofinfecting viruses could be inferred from the competition binding with OCat 0.5-10 fold concentrations of the labeling ZB. For example, cleardistinction of OC susceptible and OC resistant WSN variants was observedby treating infected cells using labeling ZB at 30 nM and competing OCat 150 nM (FIG. 13B).

MDCK cells were infected with either wild type (274H) or OC resistant(274Y) WSN viruses. At 16-20 hr post-infection, the infected cells weretreated either with 30 nM ZB or with 30 nM ZB plus 150 nM OC. Theresulting cells were decorated with streptavidin-FITC, washed andobserved using a fluorescence microscope. For flow cytometry studies,the ZB labeled cells were further labeled using anti-NP antibody,trypsinized, and then treated with PE-conjugated streptavidin plusDyLight-649 labeled anti-rabbit second antibody, both were purchasedfrom Jackson ImmunoResearch Laboratories (West Grove, Pa., USA). Afteradditional washings, the cells were analyzed by FACSCanto of BDBiosciences (San Jose, Calif., USA). For analysis of more samples, ahigh throughput method was developed using black 96-well clear bottommicroplates to culture infected cells. At 16-20 hr post-infection, cellswere labeled with 30 nM ZB or 30 nM ZB plus 150 nM OC, washed, andtreated with PE-streptavidin. The plates were then scanned with theIsocyte™ laser scanning platform of Molecular Device (Mountain View,Calif., USA) at 488 nm.

Example 26 Estimation of OC Resistant Contents Using ImmobilizedInfluenza Samples

As described with reference to FIG. 7B, the RABC assay is applicable forOC susceptibility determinations using immobilized viruses inmicro-wells. The RABC assay using immobilized viruses could also be usedto estimate the contents of OC resistant mutants in a mixed populationconsisting of both OC susceptible and resistant viruses. Viral samplesat different ratios of 274H and 274Y WSN viruses in total viral contentsat 10³, 10⁴, and 10⁵ PFU per well were immobilized to measure OCresistant ZB binding as an estimation of OC resistant contents. FIG. 14shows that the estimated OC resistant contents matched well with theexperimental 274Y viral contents that are greater than 10%. For mixedpopulations containing less than 10% resistant viruses, over-estimationof the resistant contents was observed probably due to the backgroundbinding that are higher in assays using high titer viral samples.

High binding 96-well microplates 655061 from Greiner Bio-One(Frickenhausen, Germany) were coated overnight with anti-HA antibody(Abcam) at 150 ng per well and then blotted using PBS with 3% BSA.Influenza samples were treated with 30 nM ZB or 30 nM ZB plus 150 nM OCfor 30 min and added to antibody coated wells. After incubation for 30min, the microwells were washed with 3% BSA in PBS, added with alkalinephosphatase conjugated streptavidin for 30 min, washed again with BSA inPBS and then added with the luminescence substrate Emerald-II™ accordingto the instruction of the manufacturer (Invitrogen, Carlsbad, Calif.,USA). The RLU (Relative Luminescence Unit) was read using Envision fromPerkin Elmer (Waltham, Mass., USA). Percent relative resistance wascalculated as the percent RLU determined for ZB binding measured in thepresence of OC divided by total RLU for ZB binding measured in theabsence of OC competition.

Example 27 Determination of Influenza Neuraminidase Sequences

Total RNA was extracted from influenza viruses using High Pure Viral RNAkit from Roche Diagnostics purchased at Taipei Pharma (Taipei, Taiwan).The RNA samples were reverse transcribed using random hexamers and theMMLV RTase of Toyobo Life Science Department (Tokyo, Japan) for thesynthesis of cDNA that was amplified by PCR using two primers5′-tggtcagcaagtgcwtgccatg (SEQ ID NO: 1), and 5′-gacactggaccacaactgcct(SEQ ID NO: 2) at 200 nM. The DNA products were purified and used todetermine the NA sequences.

Example 28 Rapid Detection of OC Susceptibility of Influenza Viruses onMembrane

PVDF membrane mounted on Bio-Dot SF of Bio-Rad Inc. (Bio-Rad, CA, USA)was wetted with methanol and added with 1 g anti-HA antibody (Abcam) perslot by suction. Influenza viral samples that were previously treatedfor 1 hr with either 30 nM ZB or 30 nM ZB plus 150 nM OC were introducedto neighboring slots by suction. The membranes were blotted using PBSwith 3% BSA and then incubated with alkaline phosphatase conjugatedstreptavidin from KPL (Gaithersburg, Md., USA) according to themanufacturer's instruction. After additional washing using PBS with 3%BSA, alkaline phosphatase substrate, Amresco E116 solution of AmrescoInc. (Solon, Ohio, USA) was added for color development. Visible colorusually was developed in 2 minutes and recorded by photography.

Example 29 Preparation of 293T Cells Stably Expressing Recombinant WildType (274H) and OC Resistant (274Y) Neuraminidases

The cDNA sequence of the neuraminidase gene deduced from influenzaA/Hanoi/30408/2005 (H5N1) (GeneBank: AB239126.1) was used to prepare asynthetic version of this gene by Geneart (Regensburg, Germany) andcloned into the BamH1 and XhoI restriction sites of pcDNA3.1 ofInvitrogen (Carlsbad, Calif., USA) as pcDNA3.1-NA. The NA gene wasmodified using QuickChange® XL Site-Directed Mutagenesis Kit fromAgilent Technology (La Jolla, Calif., USA) to produce OC resistant NAmutant of H274Y using a pair of primers:5′-gCTggACgCTCCCAACTACCACTACgAggAgTg-3′ (SEQ ID NO: 3) and5′-gTAgTTgggAgCgTCCAgCTCCACggAC-3 (SEQ ID NO: 4). Both the wild type(274H) and the OC resistant mutant (274Y) NA genes were validated bysequence analyses. These NA genes were used for transient NA expressionsfor wild type and OC resistant neuraminidases. They were also clonedinto the pRetro-IRES-GFP vector of Clontech (Mountain View, Calif., USA)as pRetro-NA(274H)-IRES-GFP and pRetro-NA(274Y)-IRES-GFP, respectively.Recombinant retroviruses made using these two plasmids and pGagPol, andpVSVG were used to transduce 293T cells. Stable cell lines were selectedby fluorescence-activated cell sorter for populations expressing GFPover 95% and validated for the synthesis of OC susceptible or resistantneuraminidases.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claim.

1. A method for determining the presence of oseltamivir-resistantinfluenza virus, the method comprising the steps of: (a) providing asample suspected of comprising an oseltamivir-resistant influenza-virusor virus particle; (b) contacting the sample with a binding moleculecomprising an influenza neuraminidase recognition unit (R) in thepresence and absence of oseltamivir carboxylate (OC); and (c)determining a difference in binding of the binding molecule to theinfluenza virus, or viral particle, in the presence and absence ofoseltamivir, wherein a lack of reduction in binding level by therecognition unit (R) in the presence of oseltamivir as compared to thereduction in the level of binding when contacted with aoseltamivir-sensitive influenza virus control, indicates the presence ofan oseltamivir-resistant influenza virus in the sample.
 2. The method ofclaim 1, wherein the oseltamivir-resistant influenza comprises amutation at amino acid position 274 of the neuraminidase (NA) protein ofinfluenza.
 3. The method of claim 1, wherein the influenza mutant isresistant to a neuraminidase inhibitor drug but is still sensitive tothe recognition unit (R) described in this invention.
 4. The method ofclaim 2, wherein the influenza virus containing sample is selected fromthe group consisting of mucus, saliva, respiratory secretion, throatwash, nasal wash, spinal fluid, sputum, urine, semen, sweat, feces,plasma, blood, broncheoalveolar fluid, vaginal fluid, tear fluid andtissue biopsy.
 5. The method of claim 1, wherein the binding of therecognition unit (R) to the influenza virus is competitively inhibitedby the concurrent binding by oseltamivir carboxylate.
 6. The method ofclaim 5, wherein the recognition unit (R) is selected from zanamivir, ortamiphosphor guanidine monoester, or phosphazanamivir or its monoester,and salts, esters and derivatives thereof.
 7. The method of claim 1,wherein the recognition unit (R) is coupled to a sensing unit (S) thatis detectable.
 8. The method of claim 7, wherein the recognition unit(R) is coupled to the sensing unit via a linker (L), such that thebinding molecule has the structure R-L-S.
 9. The method of claim 8,wherein the linker (L) is selected from an aliphatic chain, a triazole,a water-soluble linker, and an ethylene glycol linker.
 10. The method ofclaim 7, wherein the sensing unit is a detectable moiety selected from afluorescent label, a gold label, an enzyme label, a radioactive label, aquantum dot label, a biotin-avidin based label, and a protein label. 11.The method of claim 10, wherein the fluorescent label is selected fromthe group consisting of fluorescein, BODIPY, Alexa Fluor, Cy3, Cy5,Oregon Green, tetramethylrhodamine, Rhodamine Red, Texas Red,pyridyloxazole, benzoxadiazole derivatives, NBD halides, iodoacetamides,SBD; Lucifer Yellow, iodoacetamide; stilbene, coumarin, naphthalene,aziridine, dapoxyl, pyrene, bimanes, xanthene, cyanine, pyrene,phthalocyanine, phycobiliprotein, squarene dye, energy transfer dyecombinations, and derivatives thereof.
 12. The method of claim 1 whereinthe R-L-S molecule is selected from the group consisting of: BM 1:Zanamivir-(Triazole Linker)-Biotin

BM 2: Zanamivir-(Triazole Linker)-Fluorescein

BM 3: Zanamivir-(Ethylene Glycol Linker)-Biotin

wherein x=1 to 4, BM 4: Zanamivir-(Ethylene Glycol Linker)-Fluorescein

BM 5: (Tamiphosphor Guanidine)-(aliphatic chain Linker)-Biotin

wherein x=1 to 4, BM 6: (Tamiphosphor Guanidine)-(Ethylene GlycolLinker)-Fluorescein

BM 7: PhosphaZanamivir-(Triazole Linker)-Biotin

BM 8: PhosphaZanamivir-(Ethylene Glycol Linker)-Fluorescein

BM 9: (PhosphaZanamivir Monoester)-(Ethylene Glycol Linker)-Biotin

wherein x=1 to 4, and BM 10: (PhosphaZanamivir Monoester)-(EthyleneGlycol Linker)-Fluorescein

wherein x=1 to
 4. 13. The method of claim 1, wherein R isphosphazanamivir or a derivative thereof.
 14. The method of claim 13wherein the compound R that competitively binds oseltamivir carboxylateis a compound of formula (I),

wherein A is PO(OR)(OR⁵), wherein R and R⁵ are independently selectedfrom H, C1-C10 alkyl, aryl, aralkyl and X, wherein X is a cationiccounterion selected from the group consisting of ammonium, methylammonium, dimethylammonium, trimethylammonium, tetramethylammonium,ethanol-ammonium, dicyclohexylammonium, guanidinium, ethylenediammoniumcation, lithium cation, sodium cation, potassium cation, cesium cation,beryllium cation, magnesium cation, calcium cation, and zinc cation,wherein B is NHR⁶, NH₃ ⁺Y⁻, R⁶N(C═NH)NH₂ or R⁶N(C═NH₂ ⁺)NH₂Y⁻, whereinR⁶ represents hydrogen, C1-C10 alkyl, aryl, or aralkyl, wherein Y⁻ is aanionic counterion selected from the group consisting of chloride,bromide, iodide, acetate, trifluoroacetate, phosphate, diphosphate,nitrate, sulfate, benzenesulfonate, benzoate, salicylate,hydroxynaphthoate, fumarate, maleate, lactate, malate, succinate,tartrate, citrate, glutamate, gluconate, and stearate, wherein R¹ is CH₃or CF₃; wherein R² is H, C1-C10 alkyl or O═C—NHR⁷, wherein R⁷ representsa linker annexed with functional moiety such as biotin, fluorophore andanti-inflammatory agents, and wherein R³ and R⁴ are independentlyhydrogen, C1-C10 alkyl or O═C—R⁸, where R⁸ represents C1-C10 alkyl,aryl, or aralkyl.
 15. The method of claim 13 wherein the compound R thatcompetitively binds oseltamivir carboxylate is a compound selected fromthe group consisting of:

or a derivative thereof.